Immunopathogenesis of infection with the visceralizing Leishmania species

Human leishmaniasis is a spectral disease that includes asymptomatic self-resolving infection, localized skin lesions, and progressive visceral leishmaniasis. With some overlap, visceral and cutaneous leishmaniasis are usually caused by different species of Leishmania. This review focuses on host responses to infection with the species that cause visceral leishmaniasis, as they contrast with species causing localized cutaneous leishmaniasis. Data from experimental models document significant differences between host responses to organisms causing these diverse syndromes. The visceralizing Leishmania spp. cause localized organ-specific immune responses that are important determinants of disease outcome. Both the Leishmania species causing cutaneous and those causing visceral leishmaniasis require a Type 1 immune response to undergo cure in mouse models. However, during progressive murine infection with the visceralizing Leishmania sp., the Type 1 response is suppressed at least in part by TGF-beta and IL-10 without type 2 cytokine production. This contrasts with the cutaneous species L. major, in which a Type 2 response suppresses type 1 cytokines and leads to murine disease progression. Population and family studies are beginning to elucidate human genetic determinants predisposing to different outcomes of Leishmania infection. These studies should eventually result in a better understanding of the immunopathogenesis and the spectrum of human leishmaniasis.     

Minor histocompatibility antigen DDX3Y induces HLA-DQ5-restricted T cell responses with limited TCR-Vbeta usage both in vivo and in vitro.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.     

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

Visual inspection with acetic acid for cervical cancer screening outside of low-resource settings.

OBJECTIVES: To assess visual inspection with acetic acid (VIA) as a screening tool for use in a well-equipped health center in Peru, to evaluate VIA as an alternative or adjunct to the Papanicolaou (Pap) smear, and to determine if VIA can play a role in settings other than low-resource ones. METHODS: This was a prospective study of 1 921 asymptomatic women living in Lima, Peru, carried out in 1999 and 2000. The study was performed at a cancer center equipped with the latest-generation technology and highly trained oncologists. The women underwent a complete clinical evaluation, including a Pap smear and VIA. Participants with any positive test were referred for colposcopy and biopsy. RESULTS: More women tested positive by VIA than on the Pap smear (6.9% vs. 4.2%; P = 0.0001). There were 35 women with histologic cervical intraepithelial neoplasia grade 1 (CIN 1); of these, 15 were detected by Pap and 20 by VIA (P = 0.4). A diagnosis of CIN 2 or 3 (CIN 2-3) was confirmed in a total of 13 cases; Pap detected 5 of the cases and VIA 11 of the cases (P = 0.06). The positive predictive value for detection of CIN 2+ was 8.3% for VIA and 6.3% for Pap (P = 0.5). Most importantly, while only 2.3% of patients with a positive VIA were lost to follow-up before colposcopy, that was true for 26.3% of the women with a positive Pap smear (P < 0.0001). CONCLUSIONS: VIA is useful for detection of precursor lesions of cervical cancer not only in low-resource settings but also in well-equipped health centers and cancer centers. In these non-low-resource settings, VIA has a positive predictive value comparable to the conventional Pap smear, but it is more likely to achieve earlier diagnosis, follow-up, and treatment than cytology-based screening.     

Protective effect of γ‐tocopherol‐enriched diet on N‐methyl‐N‐nitrosourea‐induced epithelial dysplasia in rat ventral prostate

Despite recent advances in understanding the biological basis of prostate cancer (PCa), the management of this disease remains a challenge. Chemoprotective agents have been used to protect against or eradicate prostate malignancies. Here, we investigated the protective effect of γ‐tocopherol on N‐methyl‐N‐nitrosourea (MNU)‐induced epithelial dysplasia in the rat ventral prostate (VP). Thirty‐two male Wistar rats were divided into four groups (n = 8): control (CT): healthy control animals fed a standard diet; control+γ‐tocopherol (CT+γT): healthy control animals without intervention fed a γ‐tocopherol‐enriched diet (20 mg/kg); N‐methyl‐N‐nitrosourea (MNU): rats that received a single dose of MNU (30 mg/kg) plus testosterone propionate (100 mg/kg) and were fed a standard diet; and MNU+γ‐tocopherol (MNU+γT): rats that received the same treatment of MNU plus testosterone and were fed with a γ‐tocopherol‐enriched diet (20 mg/kg). After 4 months, the VPs were excised to evaluate morphology, cell proliferation and apoptosis, as well as cyclooxygenase‐2 (Cox‐2), glutathione‐S‐transferase‐pi (GST‐pi) and androgen receptor (AR) protein expression, and matrix metalloproteinase‐9 (MMP‐9) activity. An increase in the incidence of epithelial dysplasias, such as stratified epithelial hyperplasia and squamous metaplasia, in the MNU group was accompanied by augmented cell proliferation, GST‐pi and Cox‐2 immunoexpression and pro‐MMP‐9 activity. Stromal thickening and inflammatory foci were also observed. The administration of a γ‐tocopherol‐enriched diet significantly attenuated the adverse effects of MNU in the VP. The incidence of epithelial dysplasia decreased, along with the cell proliferation index, GST‐pi and Cox‐2 immunoexpression. The gelatinolytic activity of pro‐MMP‐9 returned to the levels observed for the CT group. These results suggest that γ‐tocopherol acts as a protective agent against MNU‐induced prostatic disorders in the rat ventral prostate.     

Subclinical alveolar inflammation in rheumatoid arthritis: superoxide anion, neutrophil chemotactic activity and fibronectin generation by alveolar macrophages

Interstitial lung disease (ILD) can be detected by pulmonary function testing (PFT) in 30-40% of rheumatoid arthritis (RA) patients. We assessed by bronchoalveolar lavage (BAL) the patterns of alveolitis in 21 RA patients: group 1 comprised 12 patients without evidence of ILD, and group 29 patients with clinical ILD defined by abnormal pulmonary function tests and/or chest X-ray. Cellular characteristics of BAL were studied in both groups. In addition, alveolar macrophages (AM) from patients in group 1 were isolated, and three parameters of cellular activation were studied: superoxide anion, fibronectin and neutrophil chemotactic activity generation. Total cell counts were not increased in group 1 but significantly increased in group 2 compared to controls. In group 1, 5/12 patients had elevated lymphocyte percentage (greater than 18%) suggesting subclinical lymphocyte alveolitis. In contrast, neutrophil alveolitis (greater than 4%) was found in 7/9 patients in group 2, mean percentage 12.9 +/- 4.2, compared with 1.2 +/- 6.4% in controls and 1.9 +/- 0.5% in group 1. These changes were not correlated with disease duration nor rheumatoid factor titres. Marked elevation of lymphocyte percentage was observed in patients with abnormal serum beta-2-microglobulin. Alveolar macrophages from group 1 patients released increased amounts of superoxide anion (7260 +/- 2700 vs controls 850 +/- 120 URL/5.10(5) cells), neutrophil chemotactic activity (21 +/- 4.8 vs controls 8.1 +/- 0.7 cells/HPF), and fibronectin (6.1 +/- 1.6 vs controls 1.3 +/- 0.2 ng.10(6) cells/hour). Whether or not lymphocyte alveolitis and/or AM dysfunction are pathogenic mechanisms of subsequent interstitial lung disease in patients who are still free of symptoms remains to be determined.     

The effect of gastric juice on interleukin-8 production by cystic fibrosis primary bronchial epithelial cells

CF patients are often treated with proton pump inhibitors (PPIs) to reduce acidic gastro-esophageal reflux (GER) and bronchial aspiration of duodeno-gastric contents is common in CF. We have previously demonstrated that gastric juice (GJ) from patients “on” PPI can induce interleukin-8 (IL-8) production by bronchial epithelial cells in culture. We hypothesized that such effect would be more pronounced in CF patients known to have high inflammatory susceptibility. We aimed to evaluate the effect of GJ on IL-8 production by primary bronchial epithelial cells (PBEC), derived from a CF patient and a healthy subject.MethodsPBEC obtained from one donor (normal PBEC) and one receptor (CF-PBEC) for lung transplantation were stimulated with GJ from patients “off” and “on” PPI. IL-8 levels were measured in the supernatant.ResultsGJ from patients “on” PPI provoked a significant higher IL-8 production compared to GJ from patients “off” PPI, both in normal PBEC [462 (200–1468) vs. 11 (4–28) pg/ml, p = 0.0001] as in CF-PBEC [1468 (841–2449) vs. 85 (26–131) pg/ml, p < 0.0001]. Exposure of the cells to GJ “off” PPI and “on” PPI provoked significantly higher IL-8 production in the CF-PBEC compared to the normal PBEC [“off” PPI 85 (26–131) vs. 11 (4–28) pg/ml, p = 0.01; “on” PPI 1468 (841–2449) vs. 462 (200–1468) pg/ml, p = 0.01]. Filtration (0.20 μm) of the GJ “on” PPI, to eliminate large particles and bacterial sub-products, resulted in a significant decrease of IL-8 production.ConclusionPatients with CF, treated with PPIs, have GJ with high pH and high endotoxin levels. These patients often have GER and bronchial aspiration. The aspirated material (GJ “on” PPI) has a significantly enhanced inflammatory effect on CF bronchial epithelial cells in culture. As chronic PPI treatment in CF may result in a paradoxically increased inflammatory effect in the airways, alternative anti-reflux therapies should be considered in CF.