Early appendectomy versus an interval appendectomy for appendiceal abscess in children.

We retrospectively compared the results of an early appendectomy and an interval appendectomy at a later date after initial conservative treatment in children demonstrating perforated appendicitis with a localized abscess. The preoperative conditions were similar for both groups. The early group (n = 8) showed a statistically significant longer operation time and a greater but not significant blood loss were noted while a larger number of postoperative complications were also observed. On the other hand, in the late operation group (n = 6) initial conservative management including triple antibiotic therapy proved successful without the need to drain the abscess, and thus the interval appendectomy was safely completed without any complications. There were no significant differences between the two groups with respect to length of hospital stay or medical costs. Based on these findings, we thus recommend that initial conservative treatment followed by an interval appendectomy about three months later is a useful strategy for the treatment of appendiceal abscesses in children. However, whether or not an interval appendectomy is appropriate in all patients whose inflammation is suppressed with antibiotics still needs to be clarified.

Peritoneal gamma delta T cells induced by Escherichia coli infection in mice. Correlation between Thy-1 phenotype and host minor lymphocyte-stimulating phenotype.

We found that gamma delta T cells increased in number in the peritoneal cavity after i.p. inoculation with Escherichia coli (ATCC 26) in mice. The increase of the gamma delta T cells was most prominent on day 5 after inoculation when the pathogens had been already eliminated from the hosts. Two-color flow cytometric (FCM) analysis revealed that these gamma delta T cells in infected C57BL/6 mice expressed Thy-1 Ag on their cell surface. On the other hand, gamma delta T cells induced by E. coli inoculation in C3H/He mice contained Thy-1-negative gamma delta T cells in addition to the Thy-1-positive gamma delta T cells. In both strains, irrespective of Thy-1 Ag expression, these gamma delta T cells were CD5 negative, CD44 positive, L-selectin negative, Ly-6C negative, and IL-2R low positive. Analyses of peritoneal exudate cells (PEC) from several other strains of mice after E. coli inoculation suggested that Thy-1-negative gamma delta T cells appear in mice carrying endogenous superantigen specific for V beta 3, especially mammary tumor virus-6. These findings suggest that Thy-1 Ag expression on the gamma delta T cells appearing in the peritoneal cavity after i.p. E. coli inoculation is correlated to the Mls phenotype of the host mice.

Characterization of T-cell receptor gamma delta T cells appearing at the early phase of murine Listeria monocytogenes infection.

It has been shown that T-cell receptor (TcR) gamma delta + CD4- CD8- T cells increase in number and have an important role in early protection in murine Listeria monocytogenes infection. In this report, to characterize further the phenotype of the gamma delta T cells in listeriosis, we analysed V region gene usage and in vitro antigen recognition of the TcR gamma delta T cells in the peritoneal cavity of mice at the early phase after i.p. infection with a sublethal dose of L. monocytogenes. The gamma delta T cells predominantly expressed V delta 6 which has been reported to be expressed by TcR gamma delta-bearing foetal thymocyte hybridomas specific to mycobacterial and self heat-shock protein (hsp) 60. These early appearing CD3+ CD4- CD8- T cells in Listeria-infected mice, which were reported to be TcR gamma delta T cells, increased in proportion and in size by in vitro stimulation with recombinant hsp 60 from Mycobacterium bovis and purified protein derivative from M. tuberculosis but not by stimulation with heat-killed L. monocytogenes. A 65,000 MW molecule was detected in the lysate of viable L. monocytogenes but not in the lysate of heat-killed L. monocytogenes by a monoclonal antibody (mAb) raised against mycobacterial hsp 60. These results suggest that the V delta 6-bearing peripheral gamma delta T cells are activated by recognizing listerial hsp 60 expressed by viable L. monocytogenes. The hsp 60-reactive V delta 6-bearing T cells may have an important role in protection against L. monocytogenes and other parasites that express hsp 60 at high level.

T cells expressing both L-selectin and CD44 molecules increase in number in peritoneal exudate cells and in vitro-stimulated spleen cells from mice immunized intraperitoneally with Listeria monocytogenes.

L-selectin, which was first reported as MEL-14 antigen in mice, is a type of animal lectin and expressed on lymphocytes, neutrophils and macrophages. L-selectin has been reported to be a homing receptor of lymphocytes to peripheral lymph nodes and to have an important role in initial adhesion of lymphocytes and neutrophils to endothelial cells activated by inflammatory cytokines. On the other hand, it has been reported that naive T cells express L-selectin while memory T cells and in vitro antigen-stimulated T cells lose its expression. If all memory T cells lack L-selectin, trafficking of memory T cells into inflammatory sites would be difficult. To know whether all memory T cells lack L-selectin expression, kinetics of expression of L-selectin was analysed on memory T-cell subsets, which are detected by expression of CD44, in mice after intraperitoneal immunization with a sublethal dose of viable Listeria monocytogenes. T cells expressing both L-selectin and CD44 were detected in splenocytes and peritoneal exudate cells (PEC) from untreated mice, though at low levels. L-selectin+ CD44+ T cells increased in PEC, which are known to be highly enriched in antigen-primed T cells, and reached maximum level on day 14 after immunization. Furthermore, we found increases not only of L-selectin- CD44+ but also of L-selectin+ CD44+ T cells by in vitro Listeria antigen stimulation of Listeria-immune spleen cells on day 14. These results showed that T cells expressing both L-selectin and CD44 increase after antigen stimulation in vivo and in vitro. The L-selectin+ CD44+ T cells may be a subset of memory T cells which retain their capacity of trafficking to inflammatory sites.

Effect of recombinant human granulocyte colony-stimulating factor (rh G-CSF) on murine resistance against Listeria monocytogenes.

Recombinant human granulocyte colony-stimulating factor (rh G-CSF) enhanced resistance of mice against Listeria monocytogenes (LM) as determined by survival and bacterial growth. Mice pretreated with rh G-CSF twice daily for 5 days survived better than untreated animals to the challenge with LM. Number of bacteria in peritoneal cavity (PC) and spleen was lower in treated mice than that in the control group. Rh G-CSF increased mainly polymorphonuclear cells (PMN) in blood and spleen. After LM inoculation, a larger number of PMN and monocyte-macrophages accumulated in PC and spleen of tested mice. In addition, PMN primed in vivo with rh G-CSF released more superoxide anions when stimulated with phorbol myristate acetate. The inhibition of bacterial growth in PC and spleen could be ascribed to the accumulation of phagocytic cells at the infection sites and the increased oxidative metabolism. The results provided further evidence of the important contribution of G-CSF and neutrophils, as target cells, to the host defence against the intracellular bacteria.

Difference in the functional maturation of T cells against Listeria monocytogenes in lymph nodes and spleen.

After subcutaneous immunization of mice with viable Listeria monocytogenes (LM), we evaluated the function of T cells induced in draining lymph nodes (LN) and spleen as determined by the local transfer of delayed-type hypersensitivity (DTH), acquired cellular resistance (ACR) and in vitro lymphokine production. LN cells could transfer specifically DTH but not ACR. In contrast, spleen cells from the same donor mice evoked the DTH response as well as bacterial clearance at the reaction site. Comparison of bacterial counts in spleen and in LN upon subcutaneous inoculation of mice with LM suggested that the lack of bacterial proliferation in LN underlay the failure to induce protective T cells in this lymphoid tissue. Spleen and LN T cells expressed CD4 and CD8 surface antigens equally and DTH response was solely dependent on CD4+ cells. Another major difference between LN and spleen cells was the profile of lymphokines produced in vitro. Upon the in vitro restimulation with killed Listeria, immune spleen cells produced macrophage chemotactic factor (MCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In contrast, LN cells could produce all of the measured lymphokines but not IFN-gamma. The results provided strong evidence for the dissociation of DTH and ACR. Listerial growth appeared to be the requirement for full maturation of anti-listerial immunity that may coincide with the generation of IFN-gamma-producing T cells.

A protective role of gamma/delta T cells in primary infection with Listeria monocytogenes in mice.

We have previously reported that T cells bearing T cell receptors (TCRs) of gamma/delta type appear at a relatively early stage of primary infection with Listeria monocytogenes in mice. To characterize the early-appearing gamma/delta T cells during listeriosis, we analyzed the specificity and cytokine production of the gamma/delta T cells in the peritoneal cavity in mice inoculated intraperitoneally with a sublethal dose of L. monocytogenes. The early-appearing gamma/delta T cells, most of which were of CD4-CD8- phenotype, proliferated and secreted IFN-gamma and macrophage chemotactic factor in response to purified protein derivative from Mycobacterium tuberculosis, or recombinant 65-kD heat-shock protein derived from M. bovis but not to heat-killed Listeria. To further elucidate the potential role of the gamma/delta T cells in the host-defense mechanism against primary infection with Listeria, we examined the effects of in vivo administration of monoclonal antibodies (mAbs) against TCR-gamma/delta or TCR-alpha/beta on the bacterial eradication in mice infected with Listeria. Most of alpha/beta T cells or gamma/delta T cells were depleted in the peripheral lymphoid organs at least for 12 d after an intraperitoneal injection of 200 micrograms TCR-alpha/beta mAb or 200 micrograms TCR-gamma/delta mAb, respectively. An exaggerated bacterial multiplication was evident at the early stage of listerial infection in the gamma/delta T cells-depleted mice, whereas the alpha/beta T cell-depleted mice exhibited much the same resistance level as the control mice at this stage although the resistance was severely impaired at the late stage after listerial infection.(ABSTRACT TRUNCATED AT 250 WORDS)

A dissociated induction of MCF-producing and MAF-producing T cells specific for Listeria monocytogenes in the in vitro primary culture system.

Using an in vitro primary culture system which we had previously established, the induction phase of Listeria monocytogenes-specific effector cells was analysed with respect to their abilities to produce effector lymphokines, macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF). Listeria-specific effector cells generated after in vitro culture of normal spleen cells with viable L. monocytogenes for 5 days conveyed L3T4+, Lyt-2-, Thy-1+ surface antigens and produced MCF and MAF in response to the secondary stimulation with heat-killed L. monocytogenes. The cells required for the induction of Listeria-specific effector cells, which produce effector lymphokines, MCF and MAF, were L3T4+, Lyt-2-, Thy-1+ cells. The kinetic analysis revealed that the ability of these effector cells to produce MCF was generated earlier than that to produce MAF. Furthermore, using passive transfer of cells, the effector cells producing only MCF, which were generated early in culture, conferred delayed-type hypersensitivity (DTH) alone, but MCF- and MAF-producing effector cells generated late in culture conferred sufficient levels of DTH and acquired cellular resistance (ACR). These results indicate a dissociated production of MCF and MAF by L. monocytogenes-specific T cells generated in the primary in vitro culture system.

Interleukin-1-induced promotion of T-cell differentiation in mice immunized with killed Listeria monocytogenes.

We studied the effects of administration of recombinant interleukin-1 alpha (rIL-1 alpha) to mice after immunization with killed Listeria monocytogenes cells on the promotion of the functional differentiation of T cells in vivo. Mice immunized with killed L. monocytogenes were unable to express cell-mediated immunity to specific antigen in vivo, as determined by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR), and splenic T cells obtained from such mice were unable to respond to rIL-2 and specific antigen and to produce IL-2 after antigenic restimulation in vitro. When rIL-1 alpha was given to mice after immunization with killed bacteria. T cells became capable of responding to rIL-2 and specific antigen in vitro. These functions of T cells were similar to those from mice immunized with viable listeriae. Moreover, using a local passive transfer system, it was found that effector T cells mediating DTH but not ACR to L. monocytogenes were generated in mice treated with rIL-1 alpha after immunization with killed bacteria. These T cells were able to produce macrophage chemotactic factor but not macrophage-activating factor or gamma interferon in vitro in response to stimulation with specific antigen. These results suggest that in vivo administration of rIL-1 alpha facilitates the maturation of antigen-specific T cells mediating DTH and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.

A low degree of requirement for Ia-positive macrophages and IL 2 in the induction phase of Listeria monocytogenes-specific T cells in vitro.

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, Ia antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M phi) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4+ cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2+ cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

Co-operative effect of MCF and MAF(IFN-gamma) in the protection of mice against Listeria monocytogenes.

The effects of macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF) on protection to Listeria monocytogenes were analysed using 'MCF-rich fraction' and murine recombinant interferon-gamma (rIFN-gamma). Recombinant IFN-gamma showed no macrophage chemotactic activity in the assays performed in vitro and in vivo. Although a single injection of either MCF-rich fraction or rIFN-gamma into the footpads of mice led to a significant degree of anti-listerial activity, the highest degree of protection was observed when injected together. The i.v. administration of 20,000 U rIFN-gamma did not raise significant protective activity against Listeria; however, injection with rIFN-gamma prior to that with the MCF-rich fraction into the footpad produced a higher level of protective activity than the group treated with MCF alone. In addition to MAF(IFN-gamma), MCF seems to play another important role in the full expression of protection against L. monocytogenes.

In vitro primary induction of T cells mediating delayed footpad reaction and acquired cellular resistance to Listeria monocytogenes.

We established an in vitro system generating L. monocytogenes-specific T cells primarily from unprimed spleen cells of mice. Normal spleen cells were cultured for 5 days in the presence of L. monocytogenes in vitro. Viable cells were harvested and assessed for their capacity to confer acquired cellular resistance (ACR) and delayed footpad reaction (DFR) upon local passive transfer to naive syngeneic recipient mice. When normal spleen cells were stimulated with viable L. monocytogenes, the viable cells that were recovered after 5 days of culture conferred a high level of ACR and DFR. Negative selection revealed that the effector cells obtained in primary in vitro culture were Thy 1+, L3T4+, Lyt2- cells. T cells mediating ACR could not be generated in the culture of normal spleen cells with heat-killed bacteria; however, cells mediating only DFR were generated in the presence of a large number of killed L. monocytogenes. The expression of DFR and ACR by T cells generated in this primary culture system was Listeria-specific; reactions were not observed against unrelated bacterial antigens including S. typhimurium, S. aureus, E. coli and PPD. FACS analysis of the cells in culture showed that L3T4+ and Lyt2- T cells were being enriched during culture. The primary generation of antigen-specific T cells in vitro was also possible with spleen cells from NTx mice but not with cells from nude mice, suggesting the presence of Listeria-specific precursors in NTx mice.

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.