Accuracy of diagnostic tests and a new algorithm for diagnosing cytomegalovirus colitis in inflammatory bowel diseases: a diagnostic study.

PURPOSE: The optimal method for detecting CMV colitis in patients with inflammatory bowel disease (IBD) has not been established. We wanted to investigate which diagnostic test would be most accurate when defining CMV colitis rather by the further clinical course than by using another diagnostic modality. METHODS: All consecutive patients with moderately or severely active IBD who had been tested for CMV by PCR, histology, or antigenemia assay at the two campuses CBF and CCM of the Charité - Universitätsmedizin Berlin between September 2006 and September 2009 were included in this retrospective study. During that time, in patients with a positive CMV test, immunosuppressive treatment of any kind was immediately reduced and antiviral treatment was started. This allowed identifying patients who responded to antiviral treatment and those who only responded to later escalation of immunosuppressive therapy. RESULTS: One hundred and nine patients were identified, out of whom nine were considered to have clinically relevant CMV colitis. Sensitivity and specificity were 1 and 0.94 for CMV PCR and 0.5 and 1 for pp65 antigen immunofluorescence assay from peripheral blood, 0.67 and 0.98 for immunohistochemistry, and 0.17 and 0.98 for hematoxylin-eosin staining. When using absence of leukocytosis, splenomegaly, and steroid refractory disease as clinical parameters to test for CMV colitis, blood CMV PCR and immunohistochemistry were able to exclude CMV colitis in negative patients with a 75% likelihood of positive patients to have clinically relevant CMV colitis. CONCLUSIONS: Blood-based CMV PCR together with simple clinical parameters can exclude clinically relevant CMV colitis at a high specificity.

Modulation of systemic antigen-specific immune responses by oral antigen in humans.

Oral antigen uptake can induce systemic immune responses ranging from tolerance to immunity. However, the underlying mechanisms are poorly understood, especially in humans. Here, keyhole limpet hemocyanin (KLH), a neoantigen which has been used in earlier studies of oral tolerance, was fed in a repeated low-dose and a single high-dose protocol to healthy volunteers. KLH-specific CD4(+) T-cell proliferation and cytokine production, as well as KLH-specific serum Ab and the effects of oral KLH on a subsequent parenterally induced systemic immune response, were analyzed. Repeated low-dose oral KLH alone induced antigen-specific CD4(+) T cells positive predominantly for the gut-homing receptor integrin ß7 and the cytokines IL-2 and TNF-α; some CD4(+) T cells also produced IL-4. Oral feeding of KLH accelerated a subsequent parenterally induced systemic CD4(+) T-cell response. The cytokine pattern of KLH-specific CD4(+) T cells shifted toward more IL-4- and IL-10- and less IFN-γ-, IL-2- and TNF-α-producing cells. The parenterally induced systemic KLH-specific B-cell response was accelerated and amplified by oral KLH. The impact of single high-dose oral KLH on antigen-specific immune responses was less pronounced compared with repeated low-dose oral KLH. These findings suggest that oral antigen can effectively modulate subsequently induced systemic antigen-specific immune responses. Immunomodulation by oral antigen may offer new therapeutic strategies for Th type1-mediated inflammatory diseases and for the development of vaccination strategies.

Pulmonary fibrosis after chemotherapy with oxaliplatin and 5-fluorouracil for colorectal cancer.

BACKGROUND: Chemotherapy with oxaliplatin and 5-fluorouracil (5-FU)/folinic acid is the standard first-line therapy of metastatic colorectal carcinoma and has shown activity in several other malignancies. This regimen is mostly well tolerated. Known side effects include myelosuppression, nausea/vomiting and neuropathies; acute pulmonary toxicity has only been described in very few reports. CASE REPORT: A 66-year-old male with metastatic rectal adenocarcinoma treated with 12 cycles of oxaliplatin and 5-FU/folinic acid developed bilateral pulmonary infiltrates and respiratory failure. Broad-spectrum antibiotic therapy did not improve his condition and extended microbiological diagnostics did not show an infectious etiology. Therapy with corticosteroids led to a short improvement, however the patient died 1 week after the initiation of corticosteroid treatment due to respiratory insufficiency. The clinical and histopathological data as well as the lack of an infectious cause indicate that pulmonary fibrosis was induced by oxaliplatin and 5-FU/folinic acid. CONCLUSION: This case demonstrates that treatment with oxaliplatin and 5-FU/folinic acid can cause acute pulmonary fibrosis. Even though pulmonary toxicity is rare in patients treated with this chemotherapy regimen compared to infectious pulmonary complications, it should be considered early in the clinical course of otherwise unexplained pulmonary infiltrates hopefully leading to a better outcome.

Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.

Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease.

BACKGROUND & AIMS: Regulatory CD25+ T cells (T(reg)) are effective in the prevention and down-regulation of inflammatory bowel disease (IBD) in animal models. Functional T(reg) cells are characterized by the expression of the transcription factor FOXP3 and show a CD4+ CD25(high) phenotype in humans. The aim of this study was to determine whether disease activity in IBD correlates with changes in frequency of T(reg) cells and their distribution in the intestinal mucosa. METHODS: T(reg) cells were analyzed from peripheral blood and from biopsy specimens of IBD patients, inflammatory controls, and healthy volunteers by flow cytometry (CD4+ CD25(high)), immunochemistry (FOXP3), and real-time PCR (FOXP3). Regulatory properties of purified peripheral CD4+ CD25(high) T(reg) cells were determined by their suppressive effect on the proliferation of CD4+ CD25- T cells. RESULTS: In peripheral blood, CD4+ CD25(high) T cells from IBD patients retain their suppressive activity. CD4+ CD25(high) and FOXP3+ T(reg) cells are increased during remission but decreased during active disease. This contrasts with their strong increase in peripheral blood of patients with acute diverticulitis. Different than peripheral blood, inflamed IBD mucosa contains an increased number of CD4+ CD25(high) T cells, FOXP3+ T cells, and transcripts for FOXP3 compared with noninflamed mucosa. However, the increase of FOXP3+ T cells in IBD lesions is significantly lower compared with inflammatory controls. CONCLUSIONS: The frequency of CD4+ CD25+ T(reg) cells varies with IBD activity. Active IBD is not associated with a functional defect but with a contraction of the peripheral blood T reg pool and an only moderate expansion in intestinal lesions. Thus, compensatory mechanisms, numerically, are not successfully achieved in these diseases.

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages.

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.