Cutting edge: IL-4 receptor expression by non-bone marrow-derived cells is required to expel gastrointestinal nematode parasites.

Expulsion of two gastrointestinal nematode parasites, Nippostrongylus brasiliensis and Trichinella spiralis, is similar in that both require IL-4Ralpha expression, but different in that T cells and mast cells are required for IL-4-induced expulsion of T. spiralis but not N. brasiliensis. To examine the role of IL-4Ralpha signaling in immunity to these parasites, we studied worm expulsion in chimeric mice that selectively expressed IL-4Ralpha on bone marrow-derived or non-bone marrow-derived cells. N. brasiliensis was expelled by mice that expressed IL-4Ralpha only on non-bone marrow-derived cells, but not by mice that expressed IL-4Ralpha only on bone marrow-derived cells. Although T. spiralis expulsion required IL-4Ralpha expression by both bone marrow- and non-bone marrow-derived cells, IL-4 stimulation eliminated the requirement for IL-4Ralpha expression by bone marrow-derived cells. Thus, direct IL-4Ralpha signaling of nonimmune gastrointestinal cells may be generally required to induce worm expulsion, even when mast cell and T cell responses are also required.

Thyroid alar cartilage laryngotracheal reconstruction for severe pediatric subglottic stenosis.

BACKGROUND/PURPOSE: Laryngotracheoplasty has become an accepted treatment alternative for subglottic stenosis. However, the best autogenous material for laryngotracheoplasty remains controversial. Autogenous superior thyroid alar cartilage (TAC) has been used successfully in single stage laryngotracheal reconstruction in children with subglottic stenosis. METHODS: This is a retrospective study of 6 children (mean age, 16.6 months) undergoing TAC graft laryngotracheoplasty between September 1995, and June 1999. Two children had immediate tracheal intubation for congenital subglottic stenosis. Four others had previous tracheostomy: 3 for severe postintubation subglottic stenosis and 1 for congenital subglottic stenosis. After an anterior cricoid split, a piece of TAC was sutured between the cut ends of the cricoid, with the graft perichondrium facing intraluminally. Endotracheal intubation was maintained postoperatively. RESULTS: Four children underwent successfully extubation 9 to 21 days (mean, 15.5 days) postoperatively. Two required tracheostomy, which was maintained because of severe laryngomalacia and laryngotracheobronchomalacia. One child was treated with CO2 laser because of symptomatic recurrence of the subglottic stenosis 3 weeks after the surgery; another required fundoplication for gastroesophageal reflux 12 months after laryngotracheoplasty. There were no donor site complications in any of the 6 cases. Repeat laryngoscopy and bronchoscopy showed a patent subglottic airway. All of them are without symptoms after a mean follow-up of 26 months. CONCLUSIONS: (1) This preliminary experience indicates that the TAC graft technique is a viable option for laryngotracheal reconstruction; (2) the TAC graft has significant advantages, including a single operative incision and absence of donor-site morbidity.

Alterations of intralesional and lymph node gene expression and cellular composition induced by IL-12 administration during leishmaniasis.

Changes in gene expression and cellular distribution in the lymph node and at the site of infection, the footpad, during Leishmania major infection and/or IL-12 administration were evaluated. Otherwise susceptible BALB/c mice given IL-12 are able to resolve infection. Interestingly, iNOS was not induced in the lymph node by IL-12, yet, nitric oxide is critical in the control of leishmaniasis. However, we observed an increase in iNOS at the lesion site in response to IL-12. These results reflect the importance of examining the primary site of infection. We observed no changes in inflammation at the lesion site; however, IL-12 promoted an early inflammatory response in the lymph nodes. IL-12 administration differentially affected both the local and systemic immune response to invading leishmanial parasites. IL-12 induced iNOS at the lesion site and an early granulomatous inflammation in the lymph node; therefore, we hypothesize that these are key events responsible for the resolution of disease in BALB/c mice treated with IL-12.

[Endoscopic supraglottoplasty in children with severe laryngomalacia with and without neurological impairment] / Supraglotoplastia endoscópica em crianças com laringomalacia grave com e sem doença neurológica associada.

OBJECTIVE: To describe indications and results of supraglottoplasty for severe laryngomalacia in children with or without neurological impairment. METHODS: Eight children with severe laryngomalacia submitted to endoscopic supraglottoplasty were retrospectively studied. Four had neurological impairment (male, mean age 6 years), and 4 did not present neurological problems (3 female, mean age 11.5 months). Surgery indications were respiratory distress, feeding difficulties, failure to thrive, and low oxygen saturation. Polysomnographic evaluation was carried out on the last 2 children, showing abnormal oxygen saturation, obstructive apnea, and hypoventilation. All children received preoperative antibiotics and corticosteroids. RESULTS: All children without neurological impairment had significant relief of symptoms. Children with neurological impairment had different outcome: one needed tracheotomy immediately after surgery due to edema and supraglottic granulation tissue. The other three children presented initial relief of symptoms, but subsequent follow-up showed progressive airway obstruction: one needed another endoscopic surgery 6 months later; other needed tracheotomy 7 months later. The children who were not submitted to tracheostomy presented persistent severe airway obstruction. No endoscopic surgery complication was observed. CONCLUSIONS: 1) Endoscopic supraglottoplasty is well tolerated and does not present complications when used in children; 2) Endoscopic supraglottoplasty was efficient in the treatment of children with severe laryngomalacia and in without neurological impairment; however, supraglottoplasty did not resolve airway obstruction in children with neurological impairment.

Stat6 regulation of in vivo IL-4 responses.

Although in vitro development of a Th2 response from naive CD4+ T cells is Stat6 dependent, mice immunized with a goat Ab to mouse IgD have been reported to produce a normal primary IL-4 response in Stat6-deficient mice. Experiments have now been performed with mice immunized with more conventional Ags or inoculated with nematode parasites to account for this apparent discrepancy. The ability of an immunogen to induce a primary in vivo IL-4 response in Stat6-deficient mice was found to vary directly with its ability to induce a strong type 2 cytokine-biased response in normal mice. Even immunogens, however, that induce strong primary IL-4 responses in Stat6-deficient mice induce poor memory IL-4 responses in these mice. Consistent with this, Stat6-deficient CD4+ T cells make relatively normal IL-4 responses when stimulated in vitro for 3 days with anti-CD3 and anti-CD28, but poor IL-4 responses if they are later restimulated with anti-CD3. Thus, Stat6 signaling enhances primary IL-4 responses that are made as part of a type 0 cytokine response (mixed type 1 and type 2) and is required for normal development or survival of Th2 memory cells.

Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive.

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.

Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection.

In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.

Interleukin-12 is capable of generating an antigen-specific Th1-type response in the presence of an ongoing infection-driven Th2-type response.

Previously we demonstrated that recombinant murine interleukin-12 (rmIL-12) administration can promote a primary Th1 response while suppressing the Th2 response in mice primed with 2,4, 6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The present studies examined the capacity of rmIL-12 to drive a Th1 response to TNP-KLH in the presence of an ongoing Th2-mediated disease. To establish a distinct Th2 response, we used a murine model of leishmaniasis. Susceptible BALB/c mice produce a strong Th2 response when infected with Leishmania major and develop progressive visceral disease. On day 26 postinfection, when leishmaniasis was well established, groups of mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12. Even in the presence of overt infection, TNP-KLH-plus-rmIL-12-immunized mice were still capable of generating KLH-specific gamma interferon (IFN-gamma) as well as corresponding TNP-specific immunoglobulin G2a (IgG2a) titers. In addition, the KLH-specific IL-4 was suppressed in infected mice immunized with rmIL-12. However, parasite-specific IL-4 and IgG1 production with a lack of parasite-specific IFN-gamma secretion were maintained in all infected groups of mice including those immunized with rmIL-12. These data show that despite the ongoing infection-driven Th2 response, rmIL-12 was capable of generating an antigen-specific Th1 response to an independent immunogen. Moreover, rmIL-12 administered with TNP-KLH late in infection did not alter the parasite-specific cytokine or antibody responses.

IL-13 is a key regulatory cytokine for Th2 cell-mediated pulmonary granuloma formation and IgE responses induced by Schistosoma mansoni eggs.

Schistosoma mansoni egg-induced pulmonary granuloma formation is a cell-mediated inflammatory response associated with dominant Th2-type cytokine expression, tissue eosinophilia, and high levels of serum IgE. In the present study, we show that in vivo blockade of the Th2 cytokine IL-13, using soluble IL-13R alpha2-Fc fusion protein, significantly reduced the size of pulmonary granulomas in unsensitized as well as egg-sensitized mice. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly, however, IL-13 blockade did not affect the evolving egg-induced Th2-type cytokine response. IL-4, IL-5, as well as IL-13 responses were indistinguishable in control-Fc- and soluble IL-13R alpha2-Fc fusion protein-treated animals. The smaller granulomas were also phenotypically like the control Fc-treated mice, displaying a similar eosinophil content. Additional studies in IL-4-deficient mice demonstrated that IL-13 was produced, but at much lower levels than in wild-type mice, while IL-4 expression was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development and the pulmonary eosinophilia, while it simultaneously increased IFN-gamma production. Together, these data demonstrate that IL-13 serves as an important mediator of Th2-mediated inflammation and plays a role in eliciting IgE responses triggered by schistosome eggs.

[Thoracoscopy with mediastinoscope in pediatric complicated parapneumonic effusion] / Pleuroscopia com mediastinoscópio em crianças com derrame parapneumônico complicado.

OBJECTIVE: To evaluate our experience with thoracoscopy with small mediastinoscope in complicated parapneumonic effusion in children.METHODS: From July 1995 to June 1997, seven children with complicated parapneumonic pleural effusion underwent thoracoscopy with mediastinoscope at Hospital de Clínicas de Porto Alegre. The procedure was carried out with a small mediastinoscope built in our hospital.RESULTS: There were six girls and one boy. The procedure was preformed under general anesthesia, without selective intubation. Six patients had previous intercostal tube drainage; one underwent thoracoscopy as a primary procedure. No complication was observed after the procedure. During follow-up, two children underwent pleurotomy due to residual pleural effusion with persistent fever; two others presented asymptomatic small pleural effusion.CONCLUSION: Thoracoscopy with small mediastinoscope is safe, efficient and without severe complications. It is very useful to remove loculated complicated parapneumonic effusion at fibrinopurulent stage and to enable lung expansion.

Characterization of a relatively rare class B, type 2 trypanosome variant surface glycoprotein gene.

The variant surface glycoprotein (VSG) gene of Trypanosoma brucei rhodesiense LouTat 1.5, a defined African trypanosome variant antigenic type, was cloned and sequenced. Southern blot analysis revealed 2 DNA restriction fragments in both VSG 1.5 expressor and nonexpressor populations, suggesting that there are 2 genomic copies of the VSG 1.5 gene and no expression-linked copies. Pulsed-field gel electrophoresis followed by Southern blot analysis showed that each copy of the VSG 1.5 gene exists on a separate large chromosome in both the expressor (approximately 3.5- and 4-megabase (Mb) chromosomes) and nonexpressor (approximately 4- and 5.7-Mb chromosomes) populations. Thus, VSG genes may be present on larger chromosomes than previously reported. Sequence analysis and alignments revealed that the VSG 1.5 molecule is a class B VSG with 12 cysteine residues in the N-terminus and is classified as a type 2 VSG based on C-terminus motifs. This classification shows that the VSG 1.5 molecule represents a relatively rare VSG class and type. Taken together, these studies provide additional information on VSG genes and proteins and supply the foundation for structure-function analysis of the VSG 1.5 surface antigen expressed by trypanosomes of the LouTat 1 serodeme.

The role of recombinant murine IL-12 and IFN-gamma in the pathogenesis of a murine systemic Candida albicans infection.

Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-gamma, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-gamma message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-gamma expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-gamma neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-gamma-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-gamma induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-gamma knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-gamma is required for resistance. Nonetheless, infected IFN-gamma knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-gamma and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-gamma production. Thus, we propose that although IFN-gamma can drive resistance, the overproduction of IFN-gamma during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.

Interleukin-4-dependent immunoglobulin G1 isotype switch in the presence of a polarized antigen-specific Th1-cell response to the trypanosome variant surface glycoprotein.

This study examines B-cell immunoglobulin (Ig) class-switching events in the context of parasite antigen-specific Th-cell responses in experimental African trypanosomiasis. Inbred mice were infected with Trypanosoma brucei rhodesiense, and the coordinate stimulation of Th-cell cytokine responses and B-cell responses to the trypanosome variant surface glycoprotein (VSG) was measured. The cytokines produced by T cells in response to VSG, at both the transcript and protein levels, were gamma interferon and interleukin-2 (IL-2) but not IL-4 or IL-5. Isotype profiles of antibodies specific for VSG showed that IgG1, IgG2a, and IgG3 switch responses predominated; no VSG-specific IgE responses were detected. To determine whether cryptic IL-4 responses played a role in promoting the unexpected IgG1 switch response, IL-4 knockout mice were infected; the cytokine responses and Ig isotype profiles of IL-4 knockout mice were identical to those of the wild-type control mice except for dramatically reduced IgG1 levels in response to VSG. Thus, these results revealed an IL-4-dependent component of the VSG-driven B-cell Cmu-to-Cgamma1 switch. We speculate that an IL-4 response is mediated primarily by cells other than T lymphocytes since IL-4-secreting but parasite antigen-unresponsive, "background" cells were detected in all infected mice and since infected nude mice also displayed a detectable IgG1 switch response. Overall, our results suggest that B-cell clonal stimulation, maturation, and Ig class switching in African trypanosomiasis may be partially regulated by unusual mechanisms that do not include antigen-specific Th1 or Th2 cells.

Characterization of T cell-mediated responses in nonhealing and healing Leishmania major infections in the absence of endogenous IL-4.

IL-4 drives polarized Th2 responses, and differentiating Th2 cells down-regulate their sensitivity to IL-12. Therefore, the failure of BALB/c mice to heal Leishmania major infection could be due to an IL-4-dependent biased Th2 response or to a reduced capacity of Leishmania-specific Th cells to respond to IL-12. We examined the ability of CD4+ Th cells from L. major-infected wild-type and IL-4-deficient BALB/c mice to respond to IL-12. We show that the inability of normal and IL-4-deficient BALB/c mice to heal L. major infections is due to their inability to generate effective Th1 responses and not to persistent IL-4-dominated Th2 responses. Redirection of immune responses in vivo by administration of IL-12 or anti-CD4 mAb treatment in the early phase of infection (+/-12 days) allows both normal and IL-4-deficient BALB/c mice to heal their lesions by allowing them to develop an efficient Th1 response regardless of the presence or the absence of IL-4. Finally, on a population level, Ag-specific Th cells from infected animals induced to heal display a strongly elevated response to IL-12.

[Skin lesions in progressive disseminated histoplasmosis associated with AIDS]. / Lesões cutâneas na histoplasmose disseminada progressiva associada com sida.

The protean manifestations of progressive disseminated histoplasmosis in AIDS patients (PDH/AIDS) specially the cutaneous lesions, may confuse the clinician or the pathologist. A case of PDH/AIDS diagnosed by direct and histologic examination and cultures of subcutaneous nodule aspirate and skin biopsy is reported. The requirement of special histologic and cultures procedures is emphasized. Clinical manifestations of the disease are discussed.

Characterization of T helper cell responses to the trypanosome variant surface glycoprotein.

T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.