Sarcoidosis with cryptococcal infection apparently engaging only immune-privileged body compartments: a case report.

BACKGROUND: Infection with the Cryptococcus neoformans yeast fungus is largely restricted to patients with HIV, sarcoidosis or immunosuppressive therapies. In sarcoidosis, there is intense local immune response in granuloma lesions, coupled with a paradoxical systemic anergy. An analysis of cryptococcal infection in sarcoidosis may therefore shed light on whether opportunistic pathogens preferentially engage immune-privileged tissues. CASE PRESENTATION: A 54-year-old man was admitted to our hospital after 2 months with palpitations and activity-related presyncope. A chest X-ray was normal, electrocardiography showed type-II atrioventricular-block, and there was a tentative diagnosis of myocarditis. Computed tomography reported minor hilar lymph glands and multiple nodular lesions in the lungs. Magnetic resonance imaging showed oedema and nodular structures in the heart, and fibrosis and granulomas were found in endomyocardial biopsies. The diagnosis was revised to cardiac sarcoidosis, and prednisone was initiated. In parallel, positron-emission tomography had revealed a marked uptake in the right thyroid lobe, a thyroid lobectomy was then performed, and the pathology showed a BRAF-positive papillary thyroid carcinoma. Four days postoperatively the patient developed symptoms suggestive of meningoencephalitis. Cerebrospinal fluid and blood cultures yielded growth of C. neoformans. Fungal staining of the thyroid specimen revealed cryptococcal elements in the carcinoma and in granulomas close to the tumour. Notably, there was no evidence of cryptococci in the heart sarcoid sections or in the normal thyroid parenchyma. The patient was successfully treated with antifungal agents and at the 2-year follow-up there was no evidence of thyroid cancer relapse. CONCLUSION: This sarcoidosis patient had a remarkable clinic with evidence of cryptococcal infection only in body compartments commonly regarded to be immune-privileged. The findings suggest that an opportunistic and environmentally abundant pathogen, when infecting an immunocompromised host, primarily engages immunodeficient locations such as the brain, a tumour microenvironment and some forms of granuloma.

Serological diagnostics of Lyme borreliosis: comparison of assays in twelve clinical laboratories in Northern Europe.

Lyme borreliosis (LB), caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in Europe. Laboratory diagnosis of LB is mainly based on the patients' medical history, clinical signs and symptoms in combination with detection of Borrelia-specific antibodies where indirect enzyme-linked-immunosorbent assay (ELISA) is the most widely used technique. The objective of the study was to evaluate and compare the diagnostic accuracy (sensitivities and specificities) of serological tests that are currently in use for diagnosis of LB in clinical laboratories in Northern Europe, by use of a large serum panel. The panel consisted of 195 serum samples from well-characterized and classified patients under investigation for clinically suspected LB (n = 59) including patients with Lyme neuroborreliosis, Lyme arthritis, acrodermatitis chronica atrophicans, erythema migrans or other diseases (n = 112). A total of 201 serum samples from healthy blood donors were also included. The panel (396 serum samples altogether) was sent to 12 clinical laboratories (using five different ELISA methods) as blinded for group affiliation and the laboratories were asked to perform serological analysis according to their routine procedure. The results from the study demonstrated high diagnostic concordance between the laboratories using the same diagnostic assay and lower diagnostic concordance between laboratories using different diagnostic assays. For IgG, the results were in general rather homogenous and showed an average sensitivity of 88% (range 85-91%) compared to IgM which showed lower average sensitivity of 59% (range 50-67%) and more heterogeneous results between assays and laboratories.

A man in his seventies with dyspnoea and ataxia after BCG instillation for bladder cancer. / Mann i 70-årene med tungpust og ataksi etter BCG-instillasjon mot blærekreft.

BCG instillation is a common procedure in Norwegian hospitals as part of the treatment of bladder cancer. We describe a patient who developed complications as a result of the procedure and a rare condition that proved difficult to diagnose.

Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients.

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163(+) anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.

Increased intestinal permeability in primary Sjögren's syndrome and multiple sclerosis.

There is increasing evidence suggesting a role of intestinal dysfunction in a number of autoimmune diseases. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease with a documented increased level of intestinal inflammation, whereas multiple sclerosis (MS) is an organ-specific autoimmune disease known to exhibit increased intestinal permeability. In this study we determine to what extent intestinal inflammation, analysed by a faecal calprotectin ELISA, is accompanied by altered intestinal wall permeability, as measured by a lactulose and mannitol intestinal absorption assay. Intestinal permeability was increased in both pSS and MS patients, while faecal calprotectin was elevated in pSS but normal in MS. Our findings suggest different mechanisms mediating a leaky gut in these two diseases: in pSS there is autoimmune attack directly on the intestinal wall; in MS, with autoimmunity being limited to the CNS, it may be due to a disturbed CNS regulation of enteric nerve function.

Lymphocyte and monocyte flow cytometry immunophenotyping as a diagnostic tool in uncharacteristic inflammatory disorders.

BACKGROUND: Patients with uncharacteristic inflammatory symptoms such as long-standing fatigue or pain, or a prolonged fever, constitute a diagnostic and therapeutic challenge. The aim of the present study was to determine if an extended immunophenotyping of lymphocytes and monocytes including activation markers can define disease-specific patterns, and thus provide valuable diagnostic information for these patients. METHODS: Whole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed by routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method. The immunophenotyping included the activation markers HLA-DR and CD40. Plasma levels of soluble TNF alpha receptors were analysed by ELISA. RESULTS: An informative pattern was obtained by combining two of the analysed parameters: (i), the fractions of HLA-DR-expressing CD4+ T cells and CD8+ T cells, respectively, and (ii), the level of CD40 on CD14+ CD16- monocytes. Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses. Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual variation. CONCLUSION: Immunophenotyping including the combination of the fractions of HLA-DR expressing T cell subpopulations with the level of CD40 on monocytes produces an informative pattern, differentiating between infections of bacterial and viral origin. Furthermore, a quantitative analysis of these parameters revealed the novel finding of characteristic patterns indicating a subacute bacterial infection, such as borreliosis or tuberculosis, or a mixed connective tissue disorder. The employed flow cytometric method is suitable for clinical diagnostic laboratories, and may help in the assessment of patients with uncharacteristic inflammatory symptoms.

Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer.

Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.

Human cancer, the naked mole rat and faunal turnovers.

We argue that the human evolutionary heritage with frequent adaptations through geological time to environmental change has affected a trade-off between offspring variability and cancer resistance, and thus favored cancer-prone individuals. We turn the attention to a factor setting the highly cancer-resistant naked mole rat apart from most other mammals: it has remained phenotypically largely unchanged since 30-50 million years ago. Research focusing on DNA stability mechanisms in 'living fossil' animals may help us find tools for cancer prevention and treatment.

Cytostatic drug treatment causes seeding of gene promoter methylation.

Epigenetic changes in multiple genes are emerging as an important mechanism for tumour cells to acquire resistance to chemotherapy. In the present work, we test the hypothesis that epigenetic organisation in cancer cells can be affected by cytostatic drugs. Colorectal cancer cells were cultured for several weeks in the presence of 6-thioguanine. Bisulphite sequencing of the CpG-rich promoter regions of two expressed genes showed a significantly increased frequency of methylated CpG sites in drug-treated cells, as compared with controls: 4.7% and 1.7%, respectively, for the HPRT gene; and 11.1% and 8.2% for CDX1. Essentially, all of the increase for the CDX1 gene was in a four CpG sub-region previously found to correlate with gene activity (P=0.006). This pattern of sparse promoter methylation fits with a recently proposed 'seeding' two-step mechanism leading up to gene inactivation in cancer cells. Taken together, our findings suggest activation in cancer cells of an epigenetic process enabling a tumour to generate drug-resistant variant cells.

Increased subpopulations of CD16(+) and CD56(+) blood monocytes in patients with active Crohn's disease.

BACKGROUND: Circulating monocytes may be subdivided according to the presence or absence of the Fcgamma receptor CD16 and the neural cell adhesion molecule CD56. Monocytes classified into these subpopulations are characterized by distinct phenotypic and functional features. We hypothesized that patients with active Crohn's disease differ in their peripheral monocyte subpopulations. METHODS: Using flow cytometry we investigated the expression of CD16 and CD56 on circulating monocytes in 11 patients with active Crohn's disease and 11 controls. These monocyte subpopulations were then analyzed for expression of the chemokine receptor fractalkine, CX(3)CR1, and the monocyte chemoattractant protein-1, CCR2. RESULTS: We found a median 3.7-fold increase in the number of CD16(+) monocytes related to the population with high expression of the pattern recognition receptor CD14 compared to that in the controls (P < 0.001). By studying the percentage of monocytes expressing CX(3)CR1, and their relative fluorescence intensity (RFI), we found significant differences, with both the highest percentage and the highest RFI in the CD14(low)CD16(+) subpopulation, whereas the CD14(high)CD16(+) subgroup represented an intermediate population. Inversely, CCR2 expression was highest in the populations with high expression of CD14, whereas the CD14(low)CD16(+) subpopulation showed the lowest percentage and the lowest RFI for CCR2. We found the percentage of CD14(+)CD56(+) monocytes in patients with active Crohn's disease to be increased 2.7 times compared to the controls (P = 0.011). CONCLUSIONS: These results show that subsets of peripheral monocytes with a more mature phenotype are expanded in patients with active Crohn's disease.

An inducible Ku86-degrading serine protease in human cells.

The Ku autoantigen has been implicated in a number of cellular functions including growth control, immunoglobulin gene rearrangement and DNA repair. A variant truncated form of Ku86, with an apparent molecular weight of 70 kDa, has been reported to be present in many human cell types. We have previously shown that the amount of variant Ku86 is strongly increased in human peripheral blood mononuclear cells (PBMC) by storage of blood prior to isolation of the PBMC. In this study we report that formation of variant Ku86 in protein extracts is mediated by an inducible trypsin-like serine protease with a higher concentration in the nuclear compartment, as compared with the cytoplasm. However, experiments with SDS-PAGE assay of whole cells yielded no evidence of truncated Ku86, suggesting that the protease is not active in intact cells, but is exerting a marked activity during the protein extraction procedure. Interestingly, the protease level became markedly reduced upon transfer of the cells to growth medium. Protease induction did not correlate with apoptosis, necrotic cell death or with signs of general proteolysis or cytotoxicity. Our findings have methodological implications for the interpretation of experimental Ku86 data, and suggest that this protease may play a role for cellular regulation of Ku function.

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14(+)HLA-DR(low/-)CD86(low/-)CD80(low/-)CD163(low/-)) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.

Expression of DNA-dependent protein kinase in human granulocytes.

Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNA-PK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration. In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.

A transient peak of infections during onset of rheumatoid arthritis: a 10-year prospective cohort study.

OBJECTIVES: The role of infection in rheumatoid arthritis (RA) has not been determined. We aimed to document the infectious burden and some aspects of antibacterial immunity in a large and prospective cohort study of RA patients in the early and late stages of the disease and in their relatives predisposed to RA. SETTING: Clinical and laboratory examination of all individuals enrolled in the study was performed in the Republican Clinical Hospital, Kazan, Russia. PARTICIPANTS: 376 patients with RA, 251 healthy first-degree relatives and 227 healthy controls without a family history of autoimmune disease (all females) were examined twice annually over more than 10 years. PRIMARY AND SECONDARY OUTCOME MEASURES: The following parameters were investigated: type, duration and frequency of infections, bacterial colonisation and serum levels of IgG to bacteria, serum levels of total Ig, plasma cytokine levels, granulocyte reactive oxygen species production, lysozyme activity and phagocytosis. RESULTS: There were no significant differences in infection rate between healthy controls (median 14 days/year) and RA patients (13). However, infection rates were higher (p<0.001) in healthy relatives (53) and early stage patients (62), which groups also showed heavy bacterial skin colonisation. In contrast, late stage patients had fewer infection days (12; p<0.001) than healthy controls, although bacterial colonisation was still heavy. Phagocyte function and antibacterial antibody generation, together with compensatory cytokine production, were observed to be subnormal in the healthy relatives as well as in RA patients. CONCLUSIONS: We observed a marked increase in overall infections at the time of RA onset, and signs of a defective antibacterial defence mechanism, contrasting with fewer infections in the late RA stage. It can be speculated that frequent early infections initiate a compensatory immune hyper-reactivity which reduces the infection load while stimulating the development of RA in predisposed individuals.

A high frequency of MDSCs in sepsis patients, with the granulocytic subtype dominating in gram-positive cases.

The causative microorganisms dictate the type of MDSC generated in sepsis patients, and a large proportion of PMN-MDSCs in gram-positive sepsis includes immunosuppressive myeloid blasts. MDSCs constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were identified originally in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity, and even bacterial infections. Human MDSCs are commonly divided into Mo-MDSCs and granulocytic (PMN-MDSCs) subtypes. To what extent the bona fide cancer MDSCs are representative of the proposed MDSCs found in other diseases is not well known. PMN-MDSCs have been found previously to be enriched among LDGs in density gradient-centrifuged blood. In this study, we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood compared with density gradient-centrifuged blood. We found a high frequency of typical CD14(+)HLA-DR(low) Mo-MDSCs in all sepsis patients, whereas the typical PMN-MDSCs, as well as a prominent CD14(low) PMN-MDSC-like population, appeared preferentially in gram-positive cases. The CD14(low) PMN-MDSC variant was demonstrated to suppress T cell proliferation in vitro via a ROS-dependent mechanism, to display an increased IL-10:TNF-α ratio, and to present with signs of immaturity: blast morphology and low cytokine levels. We conclude that a spectrum of cells with MDSC features is enriched in sepsis and that the microbial origin of sepsis contributes to the substantial interindividual patient variation in the MDSC pattern.