NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare tumors mainly occurring in early childhood. Our recent results showed that ectopic overexpression of human Prox1 gene, a lymphatic endothelial nuclear transcription factor, promoted an aggressive behavior in 2 murine models of KHE. This dramatic Prox1-induced phenotype prompted us to investigate immunohistochemical staining pattern of Prox1, podoplanin (D2-40), LYVE-1, and Prox1/CD34 as well as double immunofluorescent staining pattern of LYVE-1/CD31 in KHE and TA, compared with other pediatric vascular tumors. For this purpose, we examined 75 vascular lesions: KHE (n=18), TA (n=13), infantile hemangioma (n=13), pyogenic granuloma (n=18), and granulation tissue (n=13). Overall, KHE and TA shared an identical endothelial immunophenotype: the neoplastic spindle cells were Prox1, podoplanin, LYVE-1, CD31, and CD34, whereas endothelial cells within glomeruloid foci were Prox1, podoplanin, LYVE-1, CD31, and CD34. The lesional cells of all infantile hemangiomas and pyogenic granulomas were negative for Prox1 in the presence of positive internal control. These findings provide immunophenotypic evidence to support a preexisting notion that KHE and TA are closely related, if not identical. Overall, our results show, for the first time, that Prox1 is an immunohistochemical biomarker helpful in confirming the diagnosis of KHE/TA and in distinguishing it from infantile hemangioma and pyogenic granuloma.

Sarcoïdose extrapulmonaire: entité méconnue [Extrapulmonary sarcoidosis: an unrecognized disease entity?].

Sarcoidosis is a multi-systemic inflammatory disease of unknown etiology, histologically characterized by the presence of non caseating granulomas. The diagnostic suspicion relies on clinical, epidemiological, biological and radiological elements. It is confirmed by an evocative histology and by the exclusion of other granulomatous pathologies. The aim of this article is to expose some clinical manifestations of extrapulmonary sarcoidosis particularly the cardiac and abdominal involvements. A register was made on cases of sarcoidosis diagnosed in CHUV from 2000 to 2009. It demonstrates the rarity of the disease in the region of Lausanne and confirms the existence of purely extra-thoracic affections.

Effectiveness of cladribine therapy in patients with pulmonary Langerhans cell histiocytosis.

BackgroundPulmonary Langerhans cell histiocytosis (PLCH) is a rare disorder characterised by granulomatous proliferation of CD1a-positive histiocytes forming granulomas within lung parenchyma, in strong association with tobacco smoking, and which may result in chronic respiratory failure. Smoking cessation is considered to be critical in management, but has variable effects on outcome. No drug therapy has been validated. Cladribine (chlorodeoxyadenosine, 2-CDA) down-regulates histiocyte proliferation and has been successful in curbing multi-system Langerhans cell histiocytosis and isolated PLCH.Methods and patientsWe retrospectively studied 5 patients (aged 37¿55 years, 3 females) with PLCH who received 3 to 4 courses of cladribine therapy as a single agent (0.1 mg/kg per day for 5 consecutive days at monthly intervals). One patient was treated twice because of relapse at 1 year. Progressive pulmonary disease with obstructive ventilatory pattern despite smoking cessation and/or corticosteroid therapy were indications for treatment. Patients were administered oral trimethoprim/sulfamethoxazole and valaciclovir to prevent opportunistic infections. They gave written consent to receive off-label cladribine in the absence of validated treatment.ResultsFunctional class dyspnea improved with cladribine therapy in 4 out of 5 cases, and forced expiratory volume in 1 second (FEV1) increased in all cases by a mean of 387 ml (100¿920 ml), contrasting with a steady decline prior to treatment. Chest high-resolution computed tomography (HRCT) features improved with cladribine therapy in 4 patients. Hemodynamic improvement was observed in 1 patient with pre-capillary pulmonary hypertension. The results suggested a greater treatment effect in subjects with nodular lung lesions and/or thick-walled cysts on chest HRCT, with diffuse hypermetabolism of lung lesions on positron emission tomography (PET)-scan, and with progressive disease despite smoking cessation. Infectious pneumonia developed in 1 patient, with later grade 4 neutrocytopenia but without infection.DiscussionData interpretation was limited by the retrospective, uncontrolled study design and small sample size.ConclusionCladribine as a single agent may be effective therapy in patients with progressive PLCH.

Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani.

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

Interleukin-12p40 overexpression promotes interleukin-12p70 and interleukin-23 formation but does not affect bacille Calmette-Guérin and Mycobacterium tuberculosis clearance.

Interleukin (IL)-12p40, a subunit of IL-12p70 and IL-23, has previously been shown to inhibit IL-12p70 activity and interferon-gamma (IFN-gamma) production. However, recent evidence has suggested that the role of IL-12p40 is more complex. To study the contribution of IL-12p40 to immune responses against mycobacterial infections, we have used transgenic (tg) mice overexpressing IL-12p40 under the control of a major histocompatibility complex-II promoter. The IL-12p40 transgene was expressed during steady state at concentrations of 129 +/- 25 ng/ml of serum and 75 +/- 13 ng per spleen, while endogenous IL-12p40 was hardly detectable in control littermates. Bacille Calmette-Guérin (BCG) infection strongly induced the expression of IL-12p40 transgene in infected organs, and IL-12p40 monomeric and dimeric forms were identified in spleen of IL-12p40 tg mice. Excessive production of IL-12p40 resulted in a 14-fold increase in IL-12p70 serum levels in tg mice versus non-transgenic mice. IL-23 was also strongly elevated in the serum and spleens of IL-12p40 tg mice through BCG infection. While IFN-gamma and tumour necrosis factor protein levels were similar in IL-12p40 tg and non-transgenic mice, Th2 type immune responses were reduced in IL-12p40 tg mice. The number of BCG granulomas and macrophage expressing inducible nitric oxide synthase were similar in IL-12p40 tg and non-transgenic mice. IL-12p40 tg mice were as resistant as non-transgenic mice to BCG and Mycobacterium tuberculosis infections as they could efficiently control bacillary growth. These data show that high amounts of IL-12p40 promotes IL-12p70 and IL-23 formation, but that does not affect T helper 1 type immune responses and granuloma function, thus leading to normal mycobacterial clearance in infected organs.

The spectrum of opportunistic diseases complicating sarcoidosis.

Sarcoidosis is an inflammatory disease marked by a paradoxical immune status. The anergic state, which results from various immune defects, contrasts with the inflammatory formation of granulomas. Sarcoidosis patients may be at risk for opportunistic infections (OIs) and a substantial number of cases have been reported, even in untreated sarcoidosis. It is not clear how OIs in patients with sarcoidosis are different from other groups at risk. In this review, we discuss the most common OIs: mycobacterial infection (including tuberculosis), cryptococcosis, progressive multifocal leukoencephalopathy, and aspergillosis. Unlike peripheral lymphocytopenia, corticosteroids are a major risk factor for OIs but the occurrence of Ols in untreated patients suggests more complex predisposing mechanisms. Opportunistic infections presenting with extrapulmonary features are often misdiagnosed as new localizations of sarcoidosis. Aspergillomas mostly develop on fibrocystic lungs. Overall, physicians should be aware of the possible occurrence of OIs during sarcoidosis, even in untreated patients.

Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins.

BACKGROUND/AIMS: Bacillus Calmette Guerin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice. METHODS: Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice. RESULTS: iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-gamma serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately. CONCLUSIONS: These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.

Role of Toll-Like Receptor 9 Signaling in Experimental Leishmania braziliensis Infection.

Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leismaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.

Comparison between Radiographic (2-dimensional and 3-dimensional) and Histologic Findings of Periapical Lesions Treated with Apical Surgery.

INTRODUCTION: The aim of this study was to evaluate the concordance of 2- and 3-dimensional radiography and histopathology in the diagnosis of periapical lesions. METHODS: Patients were consecutively enrolled in this study provided that preoperative periapical radiography (PR) and cone-beam computed tomographic imaging of the tooth to be treated with apical surgery were performed. The periapical lesional tissue was histologically analyzed by 2 blinded examiners. The final histologic diagnosis was compared with the radiographic assessments of 4 blinded observers. The initial study material included 62 teeth in the same number of patients. RESULTS: Four lesions had to be excluded during processing, resulting in a final number of 58 evaluated cases (31 women and 27 men, mean age = 55 years). The final histologic diagnosis of the periapical lesions included 55 granulomas (94.8%) and 3 cysts (5.2%). Histologic analysis of the tissue samples from the apical lesions exhibited an almost perfect agreement between the 2 experienced investigators with an overall agreement of 94.83% (kappa = 0.8011). Radiographic assessment overestimated cysts by 28.4% (cone-beam computed tomographic imaging) and 20.7% (periapical radiography), respectively. Comparing the correlation of the radiographic diagnosis of 4 observers with the final histologic diagnosis, 2-dimensional (kappa = 0.104) and 3-dimensional imaging (kappa = 0.111) provided only minimum agreement. CONCLUSIONS: To establish a final diagnosis of an apical radiolucency, the tissue specimen should be evaluated histologically and specified as a granuloma (with/without epithelium) or a cyst. Analysis of 2-dimensional and 3-dimensional radiographic images alike results only in a tentative diagnosis that should be confirmed with biopsy.

Granulomatous Reaction to Pneumocystis jirovecii Diagnosed in a Bronchoalveolar Lavage: A Case Report.

BACKGROUND: Granulomatous reaction to Pneumocystis jirovecii is a rare but well-known pathological finding encountered in the setting of immunosuppression, HIV infection being the most common cause. It can also potentially complicate the treatment of hematological malignancies, typically when drugs lowering the count and function of lymphocytes are used. Lung biopsy is considered the gold standard for the diagnosis of granulomatous P. jirovecii pneumonia, whereas the diffuse alveolar form is usually detected cytologically in bronchoalveolar lavage (BAL). CASE: A female patient pursuing R-CHOP chemotherapy for the treatment of multiple hematological malignancies developed a rapidly progressing dyspnea. Chest CT scans revealed a worsening of a known infiltrative lung disease thought to be secondary to her chemotherapy. Alterations compatible with a drug-induced interstitial lung disease and well-formed focally necrotizing granulomas were observed on an open lung biopsy, but no microorganism was identified with special stains. Eventually, a granulomatous reaction to P. jirovecii was found in a BAL and allowed appropriate treatment with rapid improvement of the dyspnea. CONCLUSION: Because granulomas are tissue-bound structures, they are rarely described in BAL. This article describes the first reported cytological diagnosis of a granulomatous reaction to P. jirovecii and the potential diagnostic interest of such a peculiar finding.

Laparoscopic technique to perform a true Stamm gastrostomy in children.

PURPOSE: A surgical gastrostomy is mandatory in cases where a PEG is not feasible. Various minimally invasive techniques have been described, but many involve unusable materials in small children and/or have risk of disunion. We describe a technique for true Stamm gastrostomy performed by laparoscopy (LSG) with a purse string suture and four points of attachment onto the wall. METHOD: We reviewed 20 children who underwent an LSG from 2010 to 2013. After incision of the skin at the location planned for the gastrostomy, using three 3-5mm ports the stomach is fixed to the wall by three suspension stitches, which are entered and then emerged subcutaneously. A fourth stitch of attachment is used to make an award on the stomach and tie around the gastrostomy tube. RESULTS: Mean age was 4.2years, with 70% aged <2years. All children were malnourished, most often severely. All but two underwent a concomitant fundoplication. Feeding through the gastrostomy started on D0 or D1. Total feeding by gastrostomy was achieved in a mean duration of 2.9day. Mean hospital stay was 4.5days. There was no perioperative complication. Mean follow-up was 14months. Once, the balloon was accidently deflated and reinflated in the wall leading to its necrosis. Five peristomial granulomas were noticed. It was always possible to replace the tube by a gastrostomy device at least 6weeks after surgery. CONCLUSION: This new technique for true Stamm gastrostomy by laparoscopy reproduces exactly the one done by laparotomy, without special equipment. It can be made since the neonatal period, in all the circumstances when a laparoscopy is possible.