Alexander J. Gallan

Disease causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Lysosomes and phagosomes in murine cftr−/− AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr−/−, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

TRPC6 channel translocation into phagosomal membrane augments phagosomal function

Significance Historically, pulmonary infections treated with antibiotics killed bacteria while selecting for the unintended development of pathogenic resistance. One strategy to circumvent antibiotic resistance in pulmonary infection involves targeting the host phagosome and augmenting its function. To such an end, we have identified several small molecules, (R)-roscovitine and its derivatives, which restore microbicidal activity to compromised alveolar macrophages in cystic fibrosis (CF) and enhance function in non-CF cells. The compounds utilize G protein signaling pathways that mobilize TRPC-6 channels to the plasmalemma and subsequent phagosomal membrane formation that engulfs the bacterium. The plethora of GPCRs in resident pulmonary macrophages linked to ion channel function provides a rich source for potential therapeutic approaches to macrophage-mediated disease. Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

C3 glomerulopathy in adults: a distinct patient subset showing frequent association with monoclonal gammopathy and poor renal outcome

Background C3 glomerulopathy (C3G) includes both C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) and is defined by C3-dominant deposits on immunofluorescence. Dysfunction of the alternative pathway (AP) of complement is central to the pathophysiology of C3G and young patients often harbor genetic alterations of AP mediators. Recently, a link between C3G and paraproteinemia has been established. We performed this study to better characterize older patients with C3G where this association is more frequently seen. Methods Fourteen biopsies from 12 patients meeting diagnostic criteria for C3G were identified in patients > 49 years of age from 2005 to 2015 after exclusion of cases containing masked monotypic immunoglobulin deposits. Pathologic and clinical features were reviewed. Results The median age was 63.5 years and 75% of patients were male. All had renal insufficiency at presentation. Kidney biopsy showed DDD in three patients and C3GN in the remainder. Serum protein electrophoresis revealed a paraprotein in 10 patients, 8 of which had a plasma cell dyscrasia on bone marrow biopsy. A membranoproliferative pattern of glomerular injury was seen in 64% of biopsies, while mesangial proliferative and endocapillary proliferative patterns were seen less frequently. Among patients with at least 1 year of follow-up (n = 9), five were on renal replacement therapy, three showed stable (but impaired) kidney function and one demonstrated improvement. Conclusions C3G is an uncommon but important cause of kidney injury in older adults and associates with a high prevalence of paraproteinemia. In adult patients with C3G, prognosis is guarded as most patients showed either progression to end-stage kidney disease or stable but impaired kidney function.

Benign müllerian glandular inclusions in men undergoing pelvic lymph node dissection

Benign müllerian-type glandular inclusions in lymph nodes are commonly seen in women, but to our knowledge, there have only been 4 reported cases in men. Distinction of these glandular structures from metastatic adenocarcinoma is crucial for proper staging, prognosis, and treatment of the patient. We report 3 cases of benign müllerian-type glandular inclusions in men undergoing either prostatectomy or cystoprostatectomy with lymph node dissection for treatment of prostatic adenocarcinoma and/or urothelial carcinoma. None of the patients were receiving hormonal therapy. All 3 cases showed benign glands with ciliated cuboidal to columnar cells and rare secretory cells, morphologically comparable with endosalpingiosis in women. These glands were diffusely positive for PAX-8, WT-1, estrogen receptor, and progesteron receptor consistent with müllerian origin. Our study is the first to confirm müllerian origin of these glands by PAX-8 and WT-1 positivity. This finding of müllerian glands in men identical to endosalpingiosis in women supports the theory that this entity can result from müllerian metaplasia of the peritoneal mesothelium rather than displacement of tubal-type epithelium. Pathologists should also be aware that müllerian-type glands can rarely occur in men to prevent the incorrect diagnosis of metastatic adenocarcinoma involving a lymph node.

Diagnosis of Prostate Cancer with Noninvasive Estimation of Prostate Tissue Composition by Using Hybrid Multidimensional MR Imaging: A Feasibility Study

Purpose To evaluate whether compartmental analysis by using hybrid multidimensional magnetic resonance (MR) imaging can be used to diagnose prostate cancer and determine its aggressiveness. Materials and Methods Twenty-two patients with prostate cancer underwent preoperative 3.0-T MR imaging. Axial images were obtained with hybrid multidimensional MR imaging by using all combinations of echo times (47, 75, 100 msec) and b values of 0, 750, 1500 sec/mm2, resulting in a 3 × 3 array of data associated with each voxel. Volumes of the tissue components stroma, epithelium, and lumen were calculated by fitting the hybrid data to a three-compartment signal model, with distinct, paired apparent diffusion coefficient (ADC) and T2 values associated with each compartment. Volume fractions and conventional ADC and T2 were measured for regions of interest in sites of prostatectomy-verified malignancy (n = 28) and normal tissue (n = 71). Receiver operating characteristic (ROC) analysis was used to evaluate the performance of various parameters in differentiating prostate cancer from benign tissue. Results Compared with normal tissue, prostate cancer showed significantly increased fractional volumes of epithelium (23.2% ± 7.1 vs 48.8% ± 9.2, respectively) and reduced fractional volumes of lumen (26.4% ± 14.1 vs 14.0% ± 5.2) and stroma (50.5% ± 15.7 vs 37.2% ± 9.1) by using hybrid multidimensional MR imaging. The fractional volumes of tissue components show a significantly higher Spearman correlation coefficient with Gleason score (epithelium: ρ = 0.652, P = .0001; stroma: ρ = -0.439, P = .020; lumen: ρ = -0.390, P = .040) compared with traditional T2 values (ρ = -0.292, P = .132) and ADCs (ρ = -0.315, P = .102). The area under the ROC curve for differentiation of cancer from normal prostate was highest for fractional volume of epithelium (0.991), followed by fractional volumes of lumen (0.800) and stroma (0.789). Conclusion Fractional volumes of prostatic lumen, stroma, and epithelium change significantly when cancer is present. These parameters can be measured noninvasively by using hybrid multidimensional MR imaging and have the potential to improve the diagnosis of prostate cancer and determine its aggressiveness.

Candida dubliniensis Pneumonia: A Case Report and Review of Literature

Candida dubliniensis is an uncommon species of Candida which has been implicated in fungal pneumonia only very rarely. We present the case of a 75-year-old man with laryngeal cancer undergoing chemotherapy on broad-spectrum antibiotics and tuberculosis therapy with blood and endotracheal cultures positive for C. dubliniensis. Subsequent autopsy was performed with postmortem lung cultures positive for C. dubliniensis and lung histopathology demonstrating an invasive fungal infection. Molecular analysis of the lung tissue confirmed the identity of the fungi as C. dubliniensis. Since its discovery as a pathogen in the oral cavities of HIV-positive patients, C. dubliniensis has been identified in a wide spectrum of clinical scenarios and anatomic locations but manifests only rarely as pneumonia. This report represents a novel case of C. dubliniensis pneumonia confirmed by culture, histopathology, and molecular identification.

Lambda Light Chain Crystalline Cast Nephropathy and Proximal Tubulopathy

INTRODUCTION P lasma cell dyscrasias such as multiple myeloma (MM) result from clonal proliferation of plasma cells and subsequent overproduction of Igs, including free light chains. Renal dysfunction is a core manifestation of plasma cell dyscrasias. A broad range of kidney pathologies can occur, including light chain cast nephropathy, monoclonal Ig deposition disease, light chain amyloidosis, light chain proximal tubulopathy, and tubulointerstitial nephritis. Light chain proximal tubulopathy (LCPT) is a rare manifestation of monoclonal gammopathy and is characterized by the accumulation of monotypic light chains within the proximal tubule epithelial cells. The accumulated light chains frequently form crystals; however, noncrystalline forms of LCPT have been described. LCPT with crystals is associated with a kappa light chain in the vast majority of cases, which is discovered by either routine immunofluorescence (IF), IF, or immunohistochemistry after protease digestion of the paraffin-embedded tissue. LCPT without crystals can be seen with both kappaand lambda-restricted disease. When crystals are present, ultrastructural examination is often essential to the diagnosis as crystals may be easily missed by light microscopy. The diagnosis of LCPT is of great clinical importance. In up to 85% of patients, kidney biopsy showing LCPT is the initial clue to an underlying plasma cell dyscrasia before a diagnosis has been established or the disease has come to clinical attention. Therefore, the diagnosis of LCPT is crucial for appropriate patient management. We report an unusual case of LCPT in a 59-year-old woman associated with lambda light chain restriction with prominent intraluminal crystalline casts.

Collapsing Glomerulopathy in Lambda Light Chain Amyloidosis: A Report of 2 Cases

Amyloid nephropathy is an uncommon disease that frequently presents with reduced kidney function and proteinuria and, in developed nations, is most often associated with underlying paraproteinemia. The histologic appearance of glomerular amyloid deposition includes mesangial and capillary wall infiltration by an amorphous eosinophilic material, and features of endo- or extracapillary proliferation are not typically seen. Rare cases of crescentic injury have been reported in a subset of patients with amyloid nephropathy, particularly those with amyloid derived from serum amyloid A protein. Collapsing glomerulopathy, which like crescentic injury is associated with an extracapillary proliferation, has not to our knowledge been reported in the setting of amyloid nephropathy. We report 2 patients presenting with acute kidney injury and nephrotic syndrome found to have amyloid nephropathy with prominent epithelial cell hyperplasia and glomerular collapse on biopsy. This injury is likely multifactorial and related to direct podocyte injury and vascular compromise and expands further the spectrum of paraprotein-associated renal injury.

MP20-11 ACCURACY OF MULTI-PARAMETRIC MAGNETIC RESONANCE IMAGING FOR DETECTION OF PROSTATE CANCER EXTRACAPSULAR EXTENSION AND RELATION TO ITS HISTOLOGIC EXTENT

for overall detection rate of PCa and histological analysis of each Likert group (5,4,3), TB, SB and RARP samples according to Gleason score. Results determined diagnostic accuracy of biopsy samples in comparison to definitive histological diagnosis. RESULTS: 67/79 (84.1%) patients had TB positive for PCa, with a second lesion positive in 17/79 cases. Gleason score of TB and SB was equivalent in 29 (36.7%). Gleason score was higher in TB and SB in 15 (19%) and 35 (44%) cases, respectively. TB was benign in 14 patients. Gleason score at biopsy and RARP were equivalent in 49 (52.7%) cases but higher in RARP and biopsy in 10 and 34 cases, respectively. TB was PCa positive in 38/41 (92.7%) Likert 5 lesions. 2/3 remaining patients had PCa positive SB in an identical region to TB. 21/ 27 (77.8%) Likert 4 lesions had TB positive PCa, 3/6 remaining cases had PCa positive SB in an identical region to TB. TB was positive in 9/ 11 (81.8%) of Likert 3 lesions with SB negative in the remaining 2 patients. The correlation between mpMRI-TPFB-RARP was positive for 97.6% Likert 5, 88.9% Likert 4 and 85.7% Likert 3 lesions. mpMRI identified a suspicious lesion confirmed on RARP in 74 patients (93.7%). 2/5 patients with a negative correlation had a second MRI lesion that corresponded to RARP. CONCLUSIONS: The results of our analysis demonstrate the accuracy of mpMRI and the reliability of TB taken during TPFB in confirming PCa when compared to SB and definitive histological diagnosis.

Neurometabolic indicators of mitochondrial dysfunction in repetitive mild traumatic brain injury

Mild traumatic brain injury (mTBI) is a significant national health concern and there is growing evidence that repetitive mTBI (rmTBI) can cause long-term change in brain structure and function. The mitochondrion has been suggested to be involved in the mechanism of TBI. There are noninvasive methods of determining mitochondrial dysfunction through biomarkers and spectroscopy. Mitochondrial dysfunction has been implicated in a variety of neurological consequences secondary to rmTBI through activation of caspases and calpains. The purpose of this review is to examine the mechanism of mitochondrial dysfunction in rmTBI and its downstream effects on neuronal cell death, axonal injury and blood–brain barrier compromise.