Francis M. Lobo

Amprenavir-induced maculopapular exanthem followed by desensitization in a patient with late-stage human immunodeficiency virus.

BACKGROUND Amprenavir, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is approved for the treatment of HIV infection in combination with other antiretroviral agents in treatment-naive and experienced patients. Amprenavir is generally well tolerated. However, cutaneous hypersensitivity reactions to amprenavir occur in up to 28% of patients, with treatment discontinuation required in 3% of cases. OBJECTIVE To report successful desensitization to amprenavir after the occurrence of a maculopapular exanthem in an HIV-infected patient with late-stage disease and limited antiretroviral treatment options. METHODS Incremental doses of 0.025, 0.1, 0.25, 1, 2.5, 7.5, 25, 50, 100, 300, 600, and 1,200 mg of amprenavir oral solution were administered via percutaneous endoscopic gastrostomy tube at 20- to 30-minute intervals. RESULTS The patient successfully tolerated amprenavir desensitization and has continued therapy without recurrence of rash at 19 months of follow-up. CONCLUSION Desensitization may permit the continued use of amprenavir in HIV-positive patients with a history of amprenavir-induced maculopapular eruptions who have limited alternate treatment options.

Toll-Like Receptor Ligands Reverse Suppression of Contact Hypersensitivity Reactions Induced by Epicutaneous Immunization with Protein Antigen

Background: Epicutaneous (EC) immunization with protein antigens has been shown to induce antigen nonspecific suppression of subsequent T cell-dependent contact hypersensitivity (CS) reactions after active immunization. The aim of this work was to test if EC application of Toll-like receptor (TLR) ligands together with protein antigen could reverse suppression of CS. Methods: Mice were EC immunized by applying gauze patches soaked with a solution of protein antigen alone or in the presence of crude bacterial material (bacterial lysates or heat-killed bacteria) or purified TLR ligands and then tested for CS response. To test if reversal of EC-induced suppression is antigen-specific, mice were patched with TNP- or OX-substituted mouse Ig alone or together with LPS and then tested for CS with corresponding or non-cross-reacting hapten. Influence of EC immunization on cytokine production by lymph node cells was measured by ELISA. Results: EC immunization with protein antigen induces antigen nonspecific suppression that can be reversed by crude bacterial material as well as purified TLR-2, TLR-3, TLR-4, and TLR-9 ligands. The effect of TLR-4 ligand LPS was not observed in the Tlr-4 mutant C3H/HeJ mouse, indicating that this effect was dependent upon intact TLR-4 signaling. Unlike the antigen nonspecific suppression of CS by EC immunization with antigen alone, the reversal of suppression by TLR ligands was specific for the protein antigen applied in the EC protocol. Conclusions: Our results strongly suggest that EC immunization with protein antigen together with TLR ligands induces a particular antigen-specific cell population, akin to previously described contrasuppressor cells, which protects immune cells against the action of suppressor cells but have no direct influence on antigen nonspecific suppressor cells induced by antigen alone.

Epicutaneous Immunization with Protein Antigen in the Presence of TLR4 Ligand Induces TCRαβ+CD4+ T Contrasuppressor Cells That Reverse Skin-Induced Suppression of Th1-Mediated Contact Sensitivity

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-β. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRαβ CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-γ, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-γ mAb showed that IFN-γ is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

Suppression of anti-skin-allograft response by photodamaged effector cells : the modulating effects of prednisolone and cyclophosphamide

Using a murine model of skin allotransplantation, we have demonstrated previously that inhibition of specific response to alloantigen is inducible by immunization of the host with intravenously administered photoinactivated antigraft effector T cells. This hyporesponsiveness, which was demonstrated by specific inhibition of mixed leukocyte culture (MLC), inhibition of cytotoxic T lympholysis (CTL), specific suppression of the delayed type hypersensitivity (DTH) response, and prolongation of specific skin allograft survival, was adoptively transferable by CD8+ radiosensitive T lymphocytes. In this study, we extend those results to evaluate the effects of an immunosuppressive agent (prednisolone) and an alkylating drug (cyclophosphamide) on the induction of this specific suppressive cellular response. Our results reveal that the administration of prednisolone reduces the induction of the specific hyporesponsiveness to alloantigen, as demonstrated by maintenance of the DTH response to alloantigen and continued accelerated rejection of skin allografts. In contrast, the administration of cyclophosphamide augmented this specific suppressive response to alloantigen in the DTH assay and in prolongation of specific skin allograft survival. These results indicate that adjuvant immunomodulating chemotherapy alters the immune response to photoinactivated effector T cells.

Induction of a cell-transferable suppression of alloreactivity by photodamaged lymphocytes.

We have reported previously that splenocytes from BALB/c mice acutely rejecting CBA/j skin allografts were exposed to 8-methoxypsoralen (8-MOP) and ultraviolet A light and infused several times intravenously into naive BALB/c recipients; the recipients were hyporesponsive to CBA/j alloantigens in skin graft and delayed-type hypersensitivity assays, as well as in mixed leukocyte culture and cytotoxicity assays. We currently expand on this work by showing that donor-specific tolerance can be transferred adoptively to naive syngeneic animals via unfractionated splenocytes from mice rendered tolerant by the previous protocol. This suppressed response to alloantigen was transferred optimally with splenocytes taken from mice on the sixth day after the final treatment with PET cells. We further demonstrate that the cells that are adoptively transferring suppression are radiosensitive, Thy-1+, Lyt-2+, L3T4− T lymphocytes.

Broad spectrum antibiotic enrofloxacin modulates contact sensitivity through gut microbiota in a murine model

Background Medical advances in the field of infection therapy have led to an increasing use of antibiotics, which, apart from eliminating pathogens, also partially eliminate naturally existing commensal bacteria. It has become increasingly clear that less exposure to microbiota early in life may contribute to the observed rise in “immune‐mediated” diseases, including autoimmunity and allergy. Objective We sought to test whether the change of gut microbiota with the broad spectrum antibiotic enrofloxacin will modulate contact sensitivity (CS) in mice. Methods Natural gut microbiota were modified by oral treatment with enrofloxacin prior to sensitization with trinitrophenyl chloride followed by CS testing. Finally, adoptive cell transfers were performed to characterize the regulatory cells that are induced by microbiota modification. Results Oral treatment with enrofloxacin suppresses CS and production of anti–trinitrophenyl chloride IgG1 antibodies. Adoptive transfer experiments show that antibiotic administration favors induction of regulatory cells that suppress CS. Flow cytometry and adoptive transfer of purified cells show that antibiotic‐induced suppression of CS is mediated by TCR &agr;&bgr;+CD4+CD25+FoxP3+ Treg, CD19+B220+CD5+ IL‐10+, IL‐10+ Tr1, and IL‐10+ TCR &ggr;&dgr;+ cells. Treatment with the antibiotic induces dysbiosis characterized by increased proportion of Clostridium coccoides (cluster XIVa), C coccoides–Eubacterium rectale (cluster XIVab), Bacteroidetes, and Bifidobacterium spp, but decreased segmented filamentous bacteria. Transfer of antibiotic‐modified gut microbiota inhibits CS, but this response can be restored through oral transfer of control gut bacteria to antibiotic‐treated animals. Conclusions Oral treatment with a broad spectrum antibiotic modifies gut microbiota composition and promotes anti‐inflammatory response, suggesting that manipulation of gut microbiota can be a powerful tool to modulate the course of CS. Graphical abstract Figure. No Caption available.

Role of TLR ligands in epicutaneously induced contrasuppression

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-gamma production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLR-ligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

CD40 Ligand-Deficient T Cells from X-Linked Hyper-IgM Syndrome Carriers Have Intrinsic Priming Capability

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-γ and TNF-α. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.

When you hear hoof beats...do not forget the zebras.

Case presentation A 6-month-old male infant presented to his pediatrician with a 6-day history of fussiness, rhinorrhea, and shallow breathing. Before this illness, he was a healthy, full-term baby born without complications. No abnormalities were found on physical examination at the pediatrician’s office. According to parental reports, the infant’s symptoms improved until 1 to 2 days later, when the infant then developed nonbilious emesis and nonbloody diarrhea. Gastroenteritis was diagnosed, and the condition promptly resolved. The patient presented for the third time to his pediatrician with a 1-day history of increased work of breathing, lethargy, decreased feeding, and lowgrade fever. The pediatrician noted tachypnea, hypoxia, and central cyanosis and transferred the patient to a local hospital. Initial work-up included chest radiography, which revealed bilateral pneumonia. Ceftriaxone was administered. Despite supplemental oxygen and antibiotic therapy, the patient continued to have respiratory distress with impending respiratory failure and was transferred to a pediatric intensive care unit.

IgA-deficient humans exhibit gut microbiota dysbiosis despite production of compensatory IgM

Immunoglobulin A is the dominant antibody isotype found in mucosal secretions and enforces host-microbiota symbiosis in mice, yet selective IgA-deficiency (sIgAd) is the most common primary immunodeficiency in humans and is often described as asymptomatic. Here, we determined the effects of IgA deficiency on human gut microbiota composition and evaluated the possibility that secretion of IgM can compensate for a lack of secretory IgA. We used 16S rRNA gene sequencing and bacterial cell sorting to evaluate gut microbiota composition and IgA or IgM coating of the gut microbiota in 15 sIgAd subjects and 15 matched controls. Although sIgAd subjects secreted a significant amount of IgM into the intestinal lumen, this was insufficient to fully compensate for the lack of secretory IgA. Indeed, sIgAd subjects displayed an altered gut microbiota composition as compared to healthy controls, which was characterized by a trend towards decreased overall microbial diversity and significant shifts in the relative abundances of specific microbial taxa. While IgA targets a defined subset of the microbiota via high-level coating, compensatory IgM binds a broader subset of the microbiota in a less targeted manner. We conclude that IgA plays a critical and non-redundant role in controlling gut microbiota composition in humans and that secretory IgA has evolved to maintain a diverse and stable gut microbial community that promotes human health, enhances resistance to infection, and is resilient to perturbation.