The genome of an Encephalitozoon cuniculi type III strain reveals insights into the genetic diversity and mode of reproduction of a ubiquitous vertebrate pathogen.

Encephalitozoon cuniculi is a model microsporidian species with a mononucleate nucleus and a genome that has been extensively studied. To date, analyses of genome diversity have revealed the existence of four genotypes in E. cuniculi (EcI, II, III and IV). Genome sequences are available for EcI, II and III, and are all very divergent, possibly diploid and genetically homogeneous. The mechanisms that cause low genetic diversity in E. cuniculi (for example, selfing, inbreeding or a combination of both), as well as the degree of genetic variation in their natural populations, have been hard to assess because genome data have been so far gathered from laboratory-propagated strains. In this study, we aim to tackle this issue by analyzing the complete genome sequence of a natural strain of E. cuniculi isolated in 2013 from a steppe lemming. The strain belongs to the EcIII genotype and has been designated EcIII-L. The EcIII-L genome sequence harbors genomic features intermediate to known genomes of II and III lab strains, and we provide primers that differentiate the three E. cuniculi genotypes using a single PCR. Surprisingly, the EcIII-L genome is also highly homogeneous, harbors signatures of heterozygosity and also one strain-specific single-nucleotide polymorphism (SNP) that introduces a stop codon in a key meiosis gene, Spo11. Functional analyses using a heterologous system demonstrate that this SNP leads to a deficient meiosis in a model fungus. This indicates that EcIII-L meiotic machinery may be presently broken. Overall, our findings reveal previously unsuspected genome diversity in E. cuniculi, some of which appears to affect genes of primary importance for the biology of this pathogen.

GDF15 as a potential biomarker to distinguish fibrotic from non-fibrotic hypersensitivity pneumonitis.

Hypersensitivity Pneumonitis (HP) is an immune-mediated interstitial lung disease (ILD) characterized by fibrotic HP (fHP) or non-fibrotic HP (non-fHP). Fibrosis is associated with poor prognosis, emphasizing the need for biomarkers to distinguish fHP from non-fHP. This study aimed to determine the plasma levels of GDF15 in HP patients and assess its association with lung function and phenotype classification. GDF15 levels were quantified by ELISA in HP (n = 64), idiopathic pulmonary fibrosis (n = 54), and healthy control (n = 128) groups. Clinical, demographic, and functional data were obtained from medical records. High-resolution chest CT scans were used to classify HP patients into fHP and non-fHP groups. In addition, receiver operating characteristic analysis was performed to determine the cut-off point, sensitivity, and specificity. Our results revealed significantly elevated GDF15 levels in fHP compared to non-fHP (2539 ± 821 pg/ml versus 1783 ± 801 pg/ml; p = 0.009). The estimated cut-off point for plasma GDF15 levels to distinguish fHP from non-fHP was 2193.4 pg/ml (AUC 0.75). These findings suggest that GDF15 may serve as a valuable biomarker for differentiating between fHP and non-fHP, potentially indicating its involvement in lung fibrosis development in HP.

Cystic partially differentiated nephroblastoma in an 18-month-old girl: a case report.

Introduction and importance: Cystic partially differentiated nephroblastoma (CPDN) is a rare cystic tumor that affects the kidney. It has a low potential for malignancy. It usually presents as an abdominal mass. It may be difficult to confirm the diagnosis of CPDN without a histopathological study. Case presentation: The authors report a case of an 18-month-old girl with abdominal distention, which was noticed by her parents. An abdominal computed tomography scan showed a large multilocular cystic mass arising from the lower pole of the left kidney. A left total nephrectomy was performed. Immature blastemal elements without evidence of malignant cells were observed on histological analysis. Conclusion: The authors report a case of an 18-month-old girl with CPDN managed by total nephrectomy. CPDN should be considered in the differential diagnosis of patients with cystic renal lesions. The authors would also like to affirm that partial or total nephrectomy should be done in all cases of CPDN and other cystic renal tumors.

Renin is an angiotensin-independent profibrotic mediator: role in pulmonary fibrosis.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is probably the result of interplay between cytokines/chemokines and growth factors. The renin-angiotensin (Ang) system is involved, although its profibrotic effect is attributed to Ang II. However, recent studies suggest that renin, through a specific receptor, is implicated in fibrogenesis. In this study, the expression of renin and renin receptor was examined in normal and IPF lungs and fibroblasts. Normal human lung fibroblasts were stimulated with renin or transfected with renin small interfering RNA (siRNA), and the expression of transforming growth factor (TGF)-ß1 and α-1-type I collagen was analysed. Normal lungs and lung fibroblasts expressed renin, which was strongly upregulated in IPF lungs and fibroblasts (∼10-fold increase; p<0.05). Immunocytochemistry showed intense renin staining in IPF fibroblasts. Renin-stimulated lung fibroblasts displayed an increase in the expression of TGF-ß1 (mean ± sd 1.8 × 10(3) ± 0.2 × 10(3) versus 1.2 × 10(3)± 0.3 × 10(3) mRNA copies per 18S ribosomal RNA; p<0.01) and collagen (5.93 × 10(2)± 0.66 × 10(2) versus 3.28 × 10(2) ± 0.5 × 10(2); p<0.01), while knocking down renin expression using siRNA provoked a strong decrease of both molecules. These effects were independent of Ang II, since neither losartan nor captopril decreased these effects. Renin also decreased matrix metalloprotease-1 expression and induced TGF-ß1 activation (163 ± 34 versus 110 ± 15 pg active TGF-ß1 per mg total protein). These findings highlight the possible role of renin as an Ang II-independent profibrotic factor in lung fibrosis.

Genetic variants associated with severe pneumonia in A/H1N1 influenza infection.

The A/H1N1 influenza strain isolated in Mexico in 2009 caused severe pulmonary illness in a small number of exposed individuals. Our objective was to determine the influence of genetic factors on their susceptibility. We carried out a case-control association study genotyping 91 patients with confirmed severe pneumonia from A/H1N1 infection and 98 exposed but asymptomatic household contacts, using the HumanCVD BeadChip (Illumina, San Diego, CA, USA). Four risk single-nucleotide polymorphisms were significantly (p<0.0001) associated with severe pneumonia: rs1801274 (Fc fragment of immunoglobulin G, low-affinity IIA, receptor (FCGR2A) gene, chromosome 1; OR 2.68, 95% CI 1.69-4.25); rs9856661 (gene unknown, chromosome 3; OR 2.62, 95% CI 1.64-4.18); rs8070740 (RPA interacting protein (RPAIN) gene, chromosome 17; OR 2.67, 95% CI 1.63-4.39); and rs3786054 (complement component 1, q subcomponent binding protein (C1QBP) gene, chromosome 17; OR 3.13, 95% CI 1.89-5.17). All SNP associations remained significant after adjustment for sex and comorbidities. The SNPs on chromosome 17 were in linkage disequilibrium. These findings revealed that gene polymorphisms located in chromosomes 1 and 17 might influence susceptibility to development of severe pneumonia in A/H1N1 infection. Two of these SNPs are mapped within genes (FCGR2A, C1QBP) involved in the handling of immune complexes and complement activation, respectively, suggesting that these genes may confer risk due to increased activation of host immunity.

Platelet-derived growth factor in idiopathic pulmonary fibrosis.

Fibrosis is a complex process involving an inflammatory reaction, fibroblast proliferation, and abnormal accumulation of interstitial collagens. Mononuclear cells are usually present in lung fibrosis. Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. We have studied the expression of c-sis/PDGF-2 mRNA in lung tissues derived from five patients with idiopathic pulmonary fibrosis (IPF) and from four control individuals without IPF. Northern blot analysis of specimens obtained from four patients with IPF revealed the expression of the c-sis/PDGF-2 protooncogene. A control lung tissue without IPF did not express the c-sis protooncogene. In situ hybridization extended these studies demonstrating the expression of the c-sis mRNA in the five specimens with IPF but not in the four control specimens without IPF. The expression of c-sis mRNA was localized primarily in the epithelial cells. Invading alveolar macrophages also expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by the expression of PDGF-like proteins in lung specimens with IPF but not in control lung specimens. These findings demonstrate the in vivo expression of the c-sis/PDGF-2 protooncogene and the production of PDGF-like proteins in the epithelial cells and macrophages of the fibrotic tissue. This localized and sustained production of PDGF-like mitogen may constitute an important contributing factor in the abnormal fibroblast proliferation and collagen production, events associated with pulmonary fibrosis.

Innervation pattern of the pronator teres muscle.

OBJECTIVE: To determine the innervation patterns of the pronator teres muscle (PTM), which is used as a donor in muscle transfer. METHODS: This study was conducted from 2001-2006 at the Anatomy Department of the Medical Faculty of Cerrahpasa, University of Istanbul. There were 34 upper extremities of 17 fixed adult cadavers dissected. RESULTS: The classical pattern of innervation by the superior and inferior branches of the median nerve was observed in 19 of the cases (55.9%). In 4 forearms (11.8%) one branch in 10 (29.4%), 3 branches (2 humeral, 1 ulnar) and in one (2.9%), 4 branches (3 humeral, 1 ulnar) were found to be innervating the muscles. CONCLUSION: In all cases, the humeral and ulnar head of the PTM was innervated separately. These variations are of great importance during transfer of PTM.

Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection.

Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.

Leukotriene C4 upregulates collagenase expression and synthesis in human lung fibroblasts.

Leukotriene C4 (LTC4), a mediator generated by a variety of inflammatory cells, participates in several physiological and pathological processes. It has been shown that LTC4 stimulates collagen synthesis by fibroblasts, suggesting a role in collagen turnover. However, the possible effect of this mediator on collagen degradation has not been examined. In this study we explored the role of LTC4 in the modulation of fibroblast interstitial collagenase and TIMP-1. Confluent cultures of three human normal lung fibroblast cell lines, and one derived from idiopathic pulmonary fibrosis (IPF) were exposed to LTC4 0.1, 1 and 10 nM, and to IL-1 beta as positive control. Collagenase and TIMP mRNAs expression were analyzed by Northern blot followed by densitometric scanning. Immunoreactive procollagenase was detected by immunoblot, and collagenase activity was measured using [3H]collagen. Our results showed that LTC4 enhanced several-fold collagenase mRNA expression in collagenase-producing fibroblasts, and induced the expression of the enzyme mRNA in collagenase-nonproducing fibroblasts, both in normal and IPF derived cell lines. LTC4 1 nM induced the highest response. Collagenolytic activity and immunoreactive collagenase paralleled collagenase mRNA expression. Interestingly, simultaneous exposure of fibroblasts to LTC4 plus IL-1 failed to show additive effects. Moreover, in two cell lines the combination resulted in a decrease of collagenase mRNA expression compared with both mediators separately. TIMP mRNA levels were not significantly modified by LTC4, nor IL1 beta. Our findings suggest that LTC4 plays a role in the modulation of fibroblast collagenase, and it may participate in extracellular matrix remodeling during lung inflammation.

CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a lung inflammatory disorder characterized by accumulation of T lymphocytes. However, the mechanisms implicated in this process remain undefined. We examined the expression of dendritic cell (DC)-derived CC chemokine 1 (CK1)/CCL18, a chemokine putatively involved in naive T cell recruitment, in lungs from 10 patients with HP, 9 patients with idiopathic pulmonary fibrosis (IPF), and 20 healthy lungs. CCL18 was measured by real-time quantitative PCR and localized in lungs by in situ hybridization and immunohistochemistry. CCL18 expression was significantly increased in lungs affected by HP in comparison with lungs affected by IPF (2,085+/-393 vs. 1,023+/-110; P<0.05) and controls (2,085+/-393 vs. 467+/-94; P<0.01). Macrophages, DCs, and alveolar epithelial cells were the main sources of CCL18. There was a direct correlation between the levels of tissue CCL18 and the number of lymphocytes in the bronchoalveolar lavage fluids. High levels of CCL18 were detected in the subacute rather than the chronic phase of HP. These findings suggest a role for CCL18 in the pathogenesis of HP.

Preoperative risk factors associated with mortality in lung biopsy patients with interstitial lung disease.

The objective of this study was to evaluate the risk factors associated with mortality in interstitial lung disease patients. We performed a retrospective study of 722 consecutive patients submitted for lung biopsy during the 1986-1990 period. Twenty-two (3%) died within the 30 days following surgery. Forty-four patients who survived after the surgery for the same time span were randomly chosen as control group. Dyspnea at rest was present in 18/44 of surviving group (SG) and in 18/22 of the nonsurviving group (NSG) (OR 6.5, 95% CI 1.8-22.4,p = .001). Systemic diseases (i.e., diabetes, systemic arterial hypertension)were mainly present in the NSG (OR 7.2, 95% CI 2.3-22.8, p < .001). The SG displayed significantly less respiratory insufficiency with a PaO2 of 52.2 + 8.4 versus 38.5 i 9.4 mm Hg, and PaCO2 of 28.8 i 4.5 versus 38.5 +/- 9.2 mm Hg, respectively (p < .001). Likewise, the SG exhibited a PaCO2/PaO2 ratio of 0.5 - 0.1, while in the NSG it was of 1 +/- 0.4 (p < .001), showing a sensitivity of 84% and specificity of 93% for mortality. Multiple logistic regression analysis for these variables showed that log likelihood was still significant for PaCO2 > 34 mm Hg, PaO2 <48 mm Hg, and comorbid diseases. Logistic regression analysis of these three variables showed the greatest sensitivity and specificity (84 and 750/0,respectively) for prediction of mortality. However, the strongest association was found when PaCO2/PaO2 ratio was analyzed alone (OR 21,073,CI 95% 28-15,946,357, p < .005). These data suggest that PaCO2/PaO2 ratio appears to be a predictor of mortality in this subset of patients. Its prospective use has reduced early mortality after surgery less than 1% in the last decade.

Clubbing in hypersensitivity pneumonitis. Its prevalence and possible prognostic role.

To know the prevalence and prognostic significance of finger clubbing in hypersensitivity pneumonitis induced by avian antigen, this physical sign was evaluated in 82 patients who were followed up from 1 to 5 years (mean, 2.6 years). According to clinical, roentgenographic, and functional criteria, the patients were classified in one of three stages at admission as well as at least 1 year later. Digital clubbing was retrospectively recorded as present or absent by physical examination. Our results showed that 44 patients (51%) included in this study presented clubbing at the time of diagnosis. Sixteen of these patients presented with worsening of their lung disease, whereas only 5 of the 38 patients without clubbing incurred a worsening of their condition. This finding suggests that digital clubbing is frequent in pigeon breeder's disease and may help to predict clinical deterioration.

Lung alveolar epithelial cells synthesize interstitial collagenase and gelatinases A and B in vitro.

Type II pneumocytes are multifunctional alveolar epithelial cells that play a major role in the maintenance of lung structure and function. Recent evidence supports that these cells can synthesize a variety of extracellular matrix components in vitro, suggesting an active participation in connective tissue remodeling. However, their possible role in extracellular matrix degradation is unknown. In this study the production of matrix metalloproteinases (MMPs) was examined in primary cultures of rat alveolar type II pneumocytes after 2 and 7 days in culture. Under basal conditions, at both periods type II cells expressed interstitial collagenase mRNA. The immunoreactive protein was detected both in the cells and in conditioned media, and collagenolytic activity was revealed after trypsin activation. Gelatinolytic activity was detected by zymography showing a relative molecular mass of approximately 72 and 92 kDa (gelatinases A and B). Phorbol treatment increased collagenase and gelatinase activities. In addition, three alveolar epithelial cell lines were analysed for MMP production: MLE-12 (mice), L2 (rat), and A549 (human). The cell lines A549 and MLE-12 revealed collagenase and gelatinase A and B activities whereas the L2 cell line only exhibited gelatinase A activity, even after PMA induction. These findings demonstrate that alveolar epithelial cells synthesize in vitro several MMPs that confer on them the ability to degrade extracellular matrix and basement membrane components, a capacity of considerable importance for the remodeling of the stromal/epithelial interface.

Urinary thromboxane B2 as an indicator of acute rejection in lung allotransplantation.

The behavior of urinary thromboxane B2 (TXB2) during acute rejection of lung allotransplants was evaluated. Unmatched mongrel dogs were submitted to a left lung orthotopic allotransplantation (groups I and II), or a sham operation (group III). All animals had an initial significant elevation of TXB2 excretion due to surgical trauma; however, in sham-operated animals (group III) this elevation returned to basal levels after 3 days. All transplanted animals (groups I and II) had persistent TXB2 elevation with 2 important peaks on postop days 5 and 9. The elevated TXB2 excretion persisted in spite of immunosuppressive treatment with azathioprine and prednisone (group II). Rejection was followed by means of an objective grading system applied to chest roentgenograms taken on all animals. It was found that TXB2 levels correlated directly with the grade of radiographic changes seen, thus indicating degree of rejection. TXB2 can be useful as a noninvasive indicator for surveillance of lung allograft rejection.

Exponential analysis of the lung pressure-volume curve in patients with chronic pigeon-breeder's lung.

Pigeon-breeder's lung (PBL) is extremely common in Mexico City and often progresses to irreversible pulmonary fibrosis. The exponential analysis of the lung pressure-volume (PV) curve (V = A - Be-kp) has been suggested as a method to separate the lung restriction caused by inflammation from that caused by pulmonary fibrosis; a significantly decreased value for the exponential constant, k, suggests a change in the mechanical properties of the functioning lung parenchyma, while a normal value accompanied by restriction suggests subtraction of lung units without a change in the mechanical properties of the functioning units. We measured lung volumes and static PV curves in 29 patients who had persistent lung restriction following a biopsy-proven diagnosis of PBL. Mean values in the 29 subjects were as follows: age, 43 +/- 13 years; TLC, 61 +/- 15 percent of predicted; VC, 46 +/- 19 percent of predicted; and k, 55 +/- 17 percent of predicted. Twenty-four of the 29 patients had values for k that were below the 95 percent confidence level, and five had "normal" values. There was no difference in TLC and VC (percent of predicted) between those with or without a decreased value for k. Four of five patients with a normal value for k improved subsequent to diagnosis, while only one of 21 patients with a decreased k improved. We conclude that increased lung elasticity manifested by a low value for k is common in patients with chronic PBL. These results support the observation of frequent irreversible lung fibrosis in these patients. Measurements of k could prove a good prognostic indicator at the time of initial diagnosis.

Prevalence of high hematocrits in patients with interstitial lung disease in Mexico City.

Erythrocytosis, a known response to chronic hypoxemia, is considered infrequent in interstitial lung diseases. We studied the prevalence of high hematocrit (Hct) values and the relationship between Hct and SaO2 in 79 patients with chronic pigeon breeder's lung (PBL) and 34 with idiopathic pulmonary fibrosis (IPF), all of whom lived in the Mexico City metropolitan area (2,240 m above sea level). Lung biopsy was performed in 31 patients with IPF and 71 with PBL. We analyzed only one simultaneous measurement of Hct and SaO2 per patient (usually the initial measurement) before treatment. No additional cause for anemia or erythrocytosis was detected. Forty-eight percent of the patients with PBL (38/79) and 62 percent of those with IPF (21/34) had high Hct values (greater than 2 SD above mean values for Mexico City); in 14 (12.3 percent) of the 113 patients (nine with PBL and five with IPF), the Hct was above 60 percent. The Hct and SaO2 values displayed a poor correlation for the whole group: Hct = 65.7-0.16(SaO2), r = 0.24, p = 0.012. The correlation between Hct and SaO2 was nonsignificant if patients were separated by diagnosis. For an SaO2 of less than 80 percent, the slope of SaO2 vs Hct was zero. Half of our patients with PBL and IPF had Hct values that were high for the altitude. In most cases, Hct responses fell within the confidence limits reported as normal at high altitudes. We found a poor relationship between Hct and awake SaO2.

Expression of a 2.8-kb PDGF-B/c-sis transcript and synthesis of PDGF-like protein by human lung fibroblasts.

The replication of fibroblasts is thought to be controlled by exogenous growth factors mainly secreted by macrophages and epithelial cells. However, under standard culture conditions, lung fibroblasts are able to produce several growth factors, suggesting an autocrine pathway of proliferation. In this work, we examined the expression of platelet-derived growth factor (PDGF-A) and PDGF-B-messenger RNA (mRNAs) by fibroblasts derived from four human adult normal lungs and from two fibrotic lungs. Northern blot analysis showed that both normal and idiopathic pulmonary fibrosis (IPF)-derived fibroblasts expressed a 2.8 PDGF-B/c-sis mRNA. This transcript was also observed as a minor form in human osteosarcoma cell line, used as control, which predominantly expressed a 4.0-kb PDGF-B mRNA. In two fibroblast cell lines, one fibrotic and one normal, the 4.0-kb transcript was also observed but was always weaker than the 2.8-kb mRNA. PDGF-A mRNA was not detected. By immunofluorescence, lung fibroblasts exhibited intracytoplasmic PDGF-like protein. Likewise, conditioned media from normal and IPF lung fibroblasts stimulated 3H-thymidine incorporation in BALB/c-3T3 cells that was significantly inhibited by anti-PDGF antibody. These results show that in vitro, some human lung fibroblasts express PDGF-B/c-sis mRNA, mainly an alternate 2.8-kb transcript, and produce PDGF-like protein.

Lung collagenase inhibitors and spontaneous and latent collagenase activity in idiopathic pulmonary fibrosis and hypersensitivity pneumonitis.

In order to analyze the mechanisms involved in the decreased collagenolytic activity previously observed in interstitial lung fibrosis, we studied the inhibitory collagenase activity and the latent activable collagenase in lung samples from five patients with IPF, six with HP, and three control subjects. Our results showed that in both diseases, the inhibitor levels were significantly higher than in control subjects. Findings suggest that in IPF low amounts of collagenase plus excessive enzyme-inhibitors may be operating to decrease collagen catabolism. In contrast, HP lungs seem to contain adequate amounts of the enzyme but higher levels of inhibitors play a role in the abnormal degradation observed in some patients.

Lung collagen metabolism and the clinical course of hypersensitivity pneumonitis.

We studied lung collagen metabolism in 18 patients with hypersensitivity pneumonitis to determine if changes at this level could explain the different clinical courses followed by these patients. Collagen concentration, biosynthesis and degradation were measured in lung tissue samples obtained before treatment. Four patients healed, eight improved and six did not improve or worsened. All patients who healed showed an important increase in collagenolysis; patients who improved had normal or high values, but significantly less than those obtained in patients who healed. Finally, five out of the six patients who did not improve or worsened had a significant decrease in degradation. These findings support the notion that a diminution of local collagenolysis may play a role in the progression to fibrosis in some patients with hypersensitivity pneumonitis and can also be a useful tool to predict the prognosis of this disease.