Leishmania braziliensis: partial control of experimental infection by interleukin-12 p40 deficient mice

Resistance to infection by Leishmania major has been associated with the development of a Th1 type response that is dependent on the presence of interleukin 12 (IL-12). In this work the involvement of this cytokine in the response to infection by L. braziliensis, a less virulent species in the mouse model, was evaluated. Our results show that while interferon (IFN-g) deficient (-/-) mice inoculated L. braziliensis develop severe uncontrolled lesions, chronic lesions that remained under control up to 12 weeks of infection were observed in IL-12p40 -/- mice. IL 12p40 -/- mice had fewer parasites in their lesions than IFN-g-/- mice. Lymph node cells from IL-12p40 -/- were capable of producing low but consistent levels of IFN-g suggestive of its involvement in parasite control. Furthermore, as opposed to previous reports on L. major-infected animals, no switch to a Th2 response was observed in IL-12p40 -/- infected with L. braziliensis.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Further observations on clinical, histopathological, and immunological features of borderline disseminated cutaneous leishmaniasis caused by Leishmania (Leishmania) amazonensis

Leishmania (Leishmania) amazonensis has for some time been considered as the causative agent of two distinct forms of American cutaneous leishmaniasis (ACL): localized cutaneous leishmaniasis (LCL), and anergic diffuse cutaneous leishmaniasis (ADCL). Recently, a new intermediate form of disease, borderline disseminated cutaneous leishmaniasis (BDCL), was introduced into the clinical spectrum of ACL caused by this parasite, and in this paper we record the clinical, histopathological, and immunological features of eight more BDCL patients from Brazilian Amazonia, who acquired the disease in the Pará state, North Brazil. Seven of them had infections of one to two years' evolution and presented with primary skin lesions and the occurrence of metastases at periods varying from six to 12 months following appearance of the first lesion. Primary skin lesions ranged from 1-3 in number, and all had the aspect of an erythematous, infiltrated plaque, variously located on the head, arms or legs. There was lymphatic dissemination of infection, with lymph node enlargement in seven of the cases, and the delayed hypersensitivity skin-test (DTH) was negative in all eight patients prior to their treatment. After that, there was a conversion of DTH to positive in five cases re-examined. The major histopathological feature was a dermal mononuclear infiltration, with a predominance of heavily parasitized and vacuolated macrophages, together with lymphocytes and plasma cells. In one case, with similar histopathology, the patient had acquired his infection seven years previously and he presented with the largest number of disseminated cutaneous lesions. BDCL shows clinical and histopathological features which are different from those of both LCL and ADCL, and there is a good prognosis of cure which is generally not so in the case of frank ADCL.

Immunopathology of giardiasis: the role of lymphocytes in intestinal epithelial injury and malfunction

T lymphocyte-mediated pathogenesis is common to a variety of enteropathies, including giardiasis, cryptosporidiosis, bacterial enteritis, celiac's disease, food anaphylaxis, and Crohn's disease. In giardiasis as well as in these other disorders, a diffuse loss of microvillous brush border, combined or not with villus atrophy, is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water, which ultimately cause diarrheal symptoms. Other mucosal changes may include crypt hyperplasia and increased infiltration of intra-epithelial lymphocytes. Recent studies using models of giardiasis have shed new light on the immune regulation of these abnormalities. Indeed, experiments using an athymic mouse model of infection have found that these epithelial injuries were T cell-dependent. Findings from further research indicate that that the loss of brush border surface area, reduced disaccharidase activities, and increase crypt-villus ratios are mediated by CD8+ T cells, whereas both CD8+ and CD4+ small mesenteric lymph node T cells regulate the influx of intra-epithelial lymphocytes. Future investigations need to characterize the CD8+ T cell signaling cascades that ultimately lead to epithelial injury and malfunction in giardiasis and other malabsorptive disorders of the intestine.

Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Cutting edge: IL-7 regulates the peripheral pool of adult ROR gamma+ lymphoid tissue inducer cells.

During fetal life, CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch development in mice. In adult animals, CD4(+)CD3(-) cells are found in low numbers in lymphoid organs. Whether adult CD4(+)CD3(-) cells are LTi cells and are generated and maintained through cytokine signals has not been directly addressed. In this study we show that adult CD4(+)CD3(-) cells adoptively transferred into neonatal CXCR5(-/-) mice induced the formation of intestinal lymphoid tissues, demonstrating for the first time their bona fide LTi function. Increasing IL-7 availability in wild-type mice either by IL-7 transgene expression or treatment with IL-7/anti-IL-7 complexes increased adult LTi cell numbers through de novo generation from bone marrow cells and increased the survival and proliferation of LTi cells. Our observations demonstrate that adult CD4(+)lineage(-) cells are LTi cells and that the availability of IL-7 determines the size of the adult LTi cell pool.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Acute acquired toxoplasmosis: clinical-laboratorial aspects and ophthalmologic evaluation in a cohort of immunocompetent patients

Most cases of acute acquired toxoplasmosis (AAT) are oligosymptomatic and self-limited. Therefore, these infections rarely indicate treatment. Prospective studies of AAT patients are rare in the medical literature. The frequency of systemic manifestations has not been sufficiently studied. In order to search for risks factors for systemic and ocular involvement, 37 patients were submitted to a diagnostic investigative protocol. The most frequent findings were lymph node enlargement (94.6%), asthenia (86.5%), headache (70.3%), fever (67.6%) and weight loss (62.2%). Hepatomegaly and/or splenomegaly were present in 21.6% of cases (8/37). Liver transaminases were elevated in 11 patients (29.7%) and lactic dehydrogenase in 17 patients (45.9%). Anaemia was found in four patients (10.8%), leucopoenia in six patients (16.2%), lymphocytosis in 14 patients (37.8%) and thrombocytopenia in one patient (2.7%). Fundoscopic examination revealed retinochoroiditis in four patients (10.8%). No statistical association was found between any one morbidity and retinochoroiditis. Nevertheless, a significant association was found between the presence of more than eight morbidity features at evaluation and long-lasting disease. An ideal diagnostic protocol for AAT would include evidence of systemic involvement. Such a protocol could be used when planning treatment.

Primary testicular lymphoma: review

Primary testicular lymphoma is a rare form of non-Hodgkin's lymphoma. It occurs more frequently in older patients, and it is a potentially lethal disease. For early stages (I and II), the management consists of orchidectomy followed by chemotherapy combined with monoclonal antibodies directed against the CD20 antigen and scrotal radiotherapy (sanctuary site) with/or without iliac and/or paraaortic lymph node radiotherapy. For advanced stages (III and IV), chemo-immunotherapy is the treatment of choice while scrotal radiotherapy can be discussed. Both in early or advanced disease, intrathecal chemotherapy is necessary to prevent central nervous system relapse. New molecular approaches and/or more aggressive treatments are to be explored in this rare disease. To cite this journal: Oncologie 13 (2011).

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Development of T-B cell collaboration in neonatal mice.

The neonatal immune response is impaired during the first weeks after birth. To obtain a better understanding of this immaturity, we investigated the development of T cell interactions with B cells in mice. For this purpose, we analyzed the immune response to three T-dependent antigens in vivo: (i) the polyclonal antibody response induced by vaccinia virus; (ii) the production of polyclonal and specific antibodies following immunization with hapten-carrier conjugates; (iii) the mouse mammary tumor virus superantigen (sAg) response involving an increase in sAg-reactive T cells and induction of polyclonal antibody production. After vaccinia virus injection into neonates, the polyclonal antibody response was similar to that observed in adult mice. The antibody response to hapten-carrier conjugates, however, was delayed and reduced. Injection with sAg-expressing B cells from neonatal or adult mice allowed us to determine whether B cells, T cells or both were implicated in the reduced immune response. In these sAg responses, neonatal T cells were stimulated by both neonatal and adult sAg-presenting B cells but only B cells from adult mice differentiated into IgM- and IgG-secreting plasma cells in the neonatal environment in vivo. Injecting neonatal B cells into adult mice did not induce antibody production. These results demonstrate that the environment of the neonatal lymph node is able to support a T and B cell response, and that immaturity of B cells plays a key role in the reduced immune response observed in the neonate.

Randomized phase 3 trial of fluorouracil, epirubicin, and cyclophosphamide alone or followed by Paclitaxel for early breast cancer.

BACKGROUND Taxanes are among the most active drugs for the treatment of metastatic breast cancer, and, as a consequence, they have also been studied in the adjuvant setting. METHODS After breast cancer surgery, women with lymph node-positive disease were randomly assigned to treatment with fluorouracil, epirubicin, and cyclophosphamide (FEC) or with FEC followed by weekly paclitaxel (FEC-P). The primary endpoint of study-5-year disease-free survival (DFS)-was assessed by Kaplan-Meier analysis. Secondary endpoints included overall survival and analysis of the prognostic and predictive value of clinical and molecular (hormone receptors by immunohistochemistry and HER2 by fluorescence in situ hybridization) markers. Associations and interactions were assessed with a multivariable Cox proportional hazards model for DFS for the following covariates: age, menopausal status, tumor size, lymph node status, type of chemotherapy, tumor size, positive lymph nodes, HER2 status, and hormone receptor status. All statistical tests were two-sided. RESULTS Among the 1246 eligible patients, estimated rates of DFS at 5 years were 78.5% in the FEC-P arm and 72.1% in the FEC arm (difference = 6.4%, 95% confidence interval [CI] = 1.6% to 11.2%; P = .006). FEC-P treatment was associated with a 23% reduction in the risk of relapse compared with FEC treatment (146 relapses in the 614 patients in the FEC-P arm vs 193 relapses in the 632 patients in the FEC arm, hazard ratio [HR] = 0.77, 95% CI = 0.62 to 0.95; P = .022) and a 22% reduction in the risk of death (73 and 95 deaths, respectively, HR = 0.78, 95% CI = 0.57 to 1.06; P = .110). Among the 928 patients for whom tumor samples were centrally analyzed, type of chemotherapy (FEC vs FEC-P) (P = .017), number of involved axillary lymph nodes (P < .001), tumor size (P = .020), hormone receptor status (P = .004), and HER2 status (P = .006) were all associated with DFS. We found no statistically significant interaction between HER2 status and paclitaxel treatment or between hormone receptor status and paclitaxel treatment. CONCLUSIONS Among patients with operable breast cancer, FEC-P treatment statistically significantly reduced the risk of relapse compared with FEC as adjuvant therapy.

A woman with reddish nodule on the skin of the breast.

A 54-year-old woman presented a peri-areolar nodule located in the skin of the right breast. Clinical examination showed a 6 x 5 cm exophytic, lobed, ulcerated, and bleeding nodule. The patient reported that the tumor had grown gradually over a period of 3 months. The patient had been diagnosed 8 years prior to presentation with infiltrating ductal carcinoma of the right breast (pT2NO). This tumor was treated with partial mastectomy (conservative surgery) and lymph node dissection, then subsequently received 30 tangent field radiotherapy sessions to the breast for a total dose of 45 Gy. The rest of her cutaneous exam was normal. There was no family history of any similar tumor.

Selective use of postoperative neck radiotherapy in oral cavity and oropharynx cancer: a prospective clinical study.

BACKGROUND In cervical postoperative radiotherapy, the target volume is usually the same as the extension of the previous dissection. We evaluated a protocol of selective irradiation according to the risk estimated for each dissected lymph node level. METHODS Eighty patients with oral/oropharyngeal cancer were included in this prospective clinical study between 2005 and 2008. Patients underwent surgery of the primary tumor and cervical dissection, with identification of positive nodal levels, followed by selective postoperative radiotherapy. Three types of selective nodal clinical target volume (CTV) were defined: CTV0, CTV1, and CTV2, with a subclinical disease risk of <10%, 10-25%, and 25% and a prescribed radiation dose of <35 Gy, 50 Gy, and 66-70 Gy, respectively. The localization of node failure was categorized as field, marginal, or outside the irradiated field. RESULTS A consistent pattern of cervical infiltration was observed in 97% of positive dissections. Lymph node failure occurred within a high-risk irradiated area (CTV1-CTV2) in 12 patients, marginal area (CTV1/CTVO) in 1 patient, and non-irradiated low-risk area (CTV0) in 2 patients. The volume of selective lymph node irradiation was below the standard radiation volume in 33 patients (mean of 118.6 cc per patient). This decrease in irradiated volume was associated with greater treatment compliance and reduced secondary toxicity. The three-year actuarial nodal control rate was 80%. CONCLUSION This selective postoperative neck irradiation protocol was associated with a similar failure pattern to that observed after standard neck irradiation and achieved a significant reduction in target volume and secondary toxicity.