Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

OBJECTIVES: Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet-neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. METHODS: Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet-HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. RESULTS: SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. CONCLUSIONS: LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Comparison of quantitative parameters and radiomic features as inputs into machine learning models to predict the Gleason score of prostate cancer lesions.

INTRODUCTION: The classification of prostate cancer (PCa) lesions using Prostate Imaging Reporting and Data System (PI-RADS) suffers from poor inter-reader agreement. This study compared quantitative parameters or radiomic features from multiparametric magnetic resonance imaging (mpMRI) or positron emission tomography (PET), as inputs into machine learning (ML) to predict the Gleason scores (GS) of detected lesions for improved PCa lesion classification. METHODS: 20 biopsy-confirmed PCa subjects underwent imaging before radical prostatectomy. A pathologist assigned GS from tumour tissue. Two radiologists and one nuclear medicine physician delineated the lesions on the mpMR and PET images, yielding 45 lesion inputs. Seven quantitative parameters were extracted from the lesions, namely T2-weighted (T2w) image intensity, apparent diffusion coefficient (ADC), transfer constant (KTRANS), efflux rate constant (Kep), and extracellular volume ratio (Ve) from mpMR images, and SUVmean and SUVmax from PET images. Eight radiomic features were selected out of 109 radiomic features from T2w, ADC and PET images. Quantitative parameters or radiomic features, with risk factors of age, prostate-specific antigen (PSA), PSA density and volume, of 45 different lesion inputs were input in different combinations into four ML models - Decision Tree (DT), Support Vector Machine (SVM), k-Nearest-Neighbour (kNN), Ensembles model (EM). RESULTS: SUVmax yielded the highest accuracy in discriminating detected lesions. Among the 4 ML models, kNN yielded the highest accuracies of 0.929 using either quantitative parameters or radiomic features with risk factors as input. CONCLUSIONS: ML models' performance is dependent on the input combinations and risk factors further improve ML classification accuracy.

Multi-photon microscopy for the evaluation of interstitial fibrosis in extended criteria donor kidneys: A proof-of-concept study.

INTRODUCTION: To evaluate the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence in multiphoton microscopy (MPM) for the quantification of collagen/fibrosis on preimplantation biopsies of extended criteria donors (ECD). MATERIALS AND METHODS: Twenty preimplantation core biopsies were extracted from 10 donor kidney samples, of which originated from seven donors. Kidney Donor Profile Index (KDPI) and Remuzzi scores of biopsies were calculated. Collagen parameters measured included quantification by the Collagen Area Ratio in Total Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI). RESULTS: Biopsies classified with > 85% KDPI scores had significantly higher CART (p = .011) and lower CRI values (p = .025) than biopsies with ≤ 85% KDPI scores. Increase in CRI values correlated significantly with rise in recipient creatinine levels 1-year post-transplant (p = .027; 95% CI: 4.635-66.797). CONCLUSION: MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained preimplantation biopsies of donor kidneys stratified by KDPI scores. This initial evaluation found significant differences in both parameters between donor kidneys with more or less than 85% KDPI.

Tocilizumab Induces IL-10-Mediated Immune Tolerance in Invasive Candidiasis.

The existence of a hyperinflammatory state has been observed in patients with invasive fungal infections (IFI). It is being postulated whether morbidity from IFI may, in part, be a consequence of an unnecessarily prolonged or exaggerated proinflammatory immune response including interleukin 6 (IL-6) post-infection, in a host with dysregulated or compromised immunity. This, in turn, induces collateral host injury at the tissue and organ level, leading to adverse outcomes. Tocilizumab has become widely used as an immunomodulator in the treatment of inflammatory conditions. Here, we evaluated the use of tocilizumab to curb post-infective inflammatory flare in the setting of an in-vivo mouse model for invasive candidiasis. Following Candida infection, the tocilizumab-treated mice showed improved short-term survival compared with the saline-treated control mice. There was a reduced inflammatory response mounted by the host, coupled with reduced IL-6 but increased IL-10 levels. TNF-α and IFN-γ responses were not affected. Tocilizumab facilitated immune tolerance by selectively inducing IL-10, producing CD8α+ conventional dendritic cells (DCs) and peripheral T-regulatory cells, over CD11b+ conventional DCs and plasmacytoid DCs. We demonstrate here the sequelae from immunomodulatory manipulation and the basis whereby the use of monoclonal antibodies may be further explored in IFI.

Tryptophan-kynurenine ratio as a biomarker of bladder cancer.

OBJECTIVES: To investigate plasma and urinary kynurenine (KYN)-tryptophan (TRP) ratios in bladder cancer, expression of indoleamine 2,3-dioxygenase 1 (IDO1) in relation to tryptophan 2,3-dioxygenase (TDO2) in bladder tumour, and the correlation of KYN-TRP ratio with bladder tumour burden. METHODS: Metabotyping of the TRP-KYN metabolic axis was performed via a clinical case-control study. Expression of IDO1 and TDO2 was measured in human biopsied tissues. Correlational experiments between KYN-TRP ratio and bladder tumour were performed using a murine orthotopic prostate-specific antigen (PSA)-secreting MB49 bladder cancer model. RESULTS: We established for the first time that plasma TRP level was significantly decreased, while both plasma and urinary KYN-TRP ratios were significantly higher in bladder cancer patients, and expression level of IDO1 but not TDO2 was increased in human bladder tumour. We reported the positive correlation between IDO1 expression, KYN-TRP ratio, normalized PSA to creatinine, and bladder tumour burden in the murine model. CONCLUSION: Kynurenine-tryptophan ratio is a promising surveillance biomarker for bladder cancer, but would require further validation before clinical translation.

Multiple Myeloma-Associated Light Chain Amyloidosis and a Proposed Approach to Monoclonal Immunoglobulin-Associated Renal Disease.

Many challenges remain in diagnosing monoclonal immunoglobulin-associated renal disease, despite widespread application of immunofluorescence (IF) and immunohistochemistry. Here, we report a newly diagnosed case of multiple myeloma with clinical suspicion of renal amyloidosis, which had negative IF staining for kappa and lambda light chains in the glomeruli. Although laser microdissection and mass spectrometry-based proteomic analysis have emerged as important tools for amyloid typing in the literature, such facilities are still not widely available in Asia. We propose that a clinicopathological algorithm for the evaluation of organized monoclonal renal deposits, together with a combined nephrological-haematological approach, will still be adequate to generate an unequivocal diagnosis in the majority of cases.

Multiphoton Microscopic Study of the Renal Cell Carcinoma Pseudocapsule: Implications for Tumour Enucleation.

OBJECTIVE: To utilize Multiphoton Microscopy (MPM) as a novel imaging technique to characterize and quantify collagen at the Renal Cell Carcinoma Pseudocapsule, to assess for both intra-tumoral and inter-tumoral variation of collagen characteristics. MPM combines Second Harmonic Generation and Two Photon Excitation Fluorescence to image extracellular matrix architecture. METHODS: Twenty partial nephrectomy specimen tissues were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The pseudocapsules (PCs) were imaged with 2X and 20X objective at selected Regions of Interest. Corresponding clinical information was retrieved. PC thickness was determined. Collagen parameters measured included quantification by the Collagen Area Ratio, and qualitative measurements by the Collagen Fiber Density and Collagen Reticulation Index. RESULTS: The boundaries between tumor, PC and normal renal parenchyma were distinguished by MPM without need for staining. In the thickest areas of the PC, collagen content and density were quantitatively higher compared to the thinnest areas. Median Collagen Area Ratio was higher in the thickest compared to the thinnest areas of the PC (P = .01). Clear Cell RCC specimens had a consistently higher Collagen Fiber Density in both the thickest and thinnest areas compared to non-Clear Cell RCC specimens (P = .02). CONCLUSIONS: We demonstrated the ability of MPM to quantify collagen characteristics of PCs without fluorescent labeling. Tumor enucleation for Renal Cell Carcinoma along its PC remains debatable with regards to oncological safety. Even with a complete and intact PC, the PC is not a homogenous structure, and varies in its thickness and its collagen characteristics within, and between tumors.

Deletion of CD137 Ligand Exacerbates Renal and Cutaneous but Alleviates Cerebral Manifestations in Lupus.

The CD137-CD137 ligand (CD137L) costimulatory system is a critical immune checkpoint with pathophysiological implications in autoimmunity. In this study, we investigated the role of CD137L-mediated costimulation on renal, cutaneous and cerebral manifestations in lupus and the underlying immunological mechanism. Lupus-prone C57BL/6lpr-/- (B6.lpr) mice were crossed to C57BL/6.CD137L-/- mice to obtain CD137L-deficient B6.lpr [double knock out (DKO)] mice. We investigated the extent of survival, glomerulonephritis, skin lesions, cerebral demyelination, immune deviation and long-term synaptic plasticity among the two mouse groups. Cytokine levels, frequency of splenic leukocyte subsets and phenotypes were compared between DKO, B6.lpr and B6.WT mice. A 22 month observation of 226 DKO and 137 B6.lpr mice demonstrated significantly more frequent proliferative glomerulonephritis, larger skin lesions and shorter survival in DKO than in B6.lpr mice. Conversely, microglial activation and cerebral demyelination were less pronounced while long-term synaptic plasticity, was superior in DKO mice. Splenic Th17 cells were significantly higher in DKO than in B6.lpr and B6.WT mice while Th1 and Th2 cell frequencies were comparable between DKO and B6.lpr mice. IL-10 and IL-17 expression by T cells was not affected but there were fewer IL-10-producing myeloid (CD11b+) cells, and also lower serum IL-10 levels in DKO than in B6.lpr mice. The absence of CD137L causes an immune deviation toward Th17, fewer IL-10-producing CD11b+ cells and reduced serum IL-10 levels which potentially explain the more severe lupus in DKO mice while leading to reduced microglia activation, lesser cerebral damage and less severe neurological deficits.

TLR7 Protein Expression in Mild and Severe Lupus-Prone Models Is Regulated in a Leukocyte, Genetic, and IRAK4 Dependent Manner.

The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the Sle1 and Sle1-TLR7 transgenic (Sle1Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.

Severe ischemic cytomegalovirus proctocolitis with multiple perforation.

Cytomegalovirus (CMV) typically causes gastrointestinal infections in immunocompetent patients. Colonic perforations secondary to CMV are exceeding rare. We describe a 88-year-old male presenting with a week-long history of intractable abdominal discomfort, bloating, nausea and diarrhea. Flexible sigmoidoscopy revealed significant ulceration with yellowish slough. Emergency surgery was performed subsequently in view of multiple perforations in the rectosigmoid junction. CMV gastrointestinal infections demonstrated an ischemic process secondary to vasculitis, which accelerated the pathway to colonic perforation. CMV gastrointestinal infection should be considered as a differential diagnosis in patients with colonoscopy findings similar to ischemic colitis and Clostridium difficile infections.

Solitary vertebral metastasis of primary clear cell carcinoma of the liver: a case report and review of literature.

Primary clear cell carcinoma of liver (PCCCL) is an uncommon variant of primary hepatocellular carcinoma. Though the literature describes a better prognosis in relation to the proportion of clear cells in the tumour when compared to the other variants, there is no general consensus in the management due to its rarity and unclear clinicopathological and prognostic factors. There is dearth of evidence with regard to the metastasizing nature of PCCCL and its management. In addition, the management of recurrent spinal tumours both primary and metastatic is not clear as the available evidence is mostly based on case reports. We describe an unusual presentation of PCCCL with solitary spinal metastasis and further complicated by tumour recurrence in a 71-year-old male. Such presentation has never been described before. He presented with low back pain and incomplete neurological deficits involving both lower limbs. On detailed evaluation, he was found to have a solitary metastasis at L3 vertebra secondary to PCCCL. He underwent radical excision of tumour and reconstruction for the solitary metastasis at L3 vertebral body and trans arterial chemo embolisation (TACE) for the hepatic lesion. Pt was asymptomatic until 9 months post operatively when he developed tumour recurrence at L3 vertebra. Patient subsequently underwent 2 stage palliative surgery followed by radiotherapy and chemotherapy. At his latest follow-up (1 year), the patient's overall general condition has improved with residual neurological deficits in the lower limb. PCCCL is a rare type of hepatocellular carcinoma which can present as "solitary metastasis" to the spine. Although the literature suggests a good prognosis for this histological type, this case did not have a good outcome. In addition to providing information for the management of similar cases in the future, this case report highlights that every patient has to be managed on a case-by-case basis.

Bimodal Influence of Vitamin D in Host Response to Systemic Candida Infection-Vitamin D Dose Matters.

Vitamin D level is linked to susceptibility to infections, but its relevance in candidemia is unknown. We aimed to investigate the in vivo sequelae of vitamin D3 supplementation in systemic Candida infection. Implicating the role of vitamin D in Candida infections, we showed that candidemic patients had significantly lower 25-OHD concentrations. Candida-infected mice treated with low-dose 1,25(OH)2D3 had reduced fungal burden and better survival relative to untreated mice. Conversely, higher 1,25(OH)2D3 doses led to poor outcomes. Mechanistically, low-dose 1,25(OH)2D3 induced proinflammatory immune responses. This was mediated through suppression of SOCS3 and induction of vitamin D receptor binding with the vitamin D-response elements in the promoter of the gene encoding interferon γ. These beneficial effects were negated with higher vitamin D3 doses. While the antiinflammatory effects of vitamin D3 are well described, we found that, conversely, lower doses conferred proinflammatory benefits in Candida infection. Our study highlights caution against extreme deviations of vitamin D levels during infections.

Molecular profiling reveals a tumor-promoting phenotype of monocytes and macrophages in human cancer progression.

Monocytes and macrophages are major components of the tumor microenvironment, but their contributions to human cancer are poorly understood. We used molecular profiling combined with functional assays to investigate the role of these cells in human renal cell carcinoma (RCC). Blood monocytes from RCC patients displayed a tumor-promoting transcriptional profile that supported functions like angiogenesis and invasion. Induction of this protumor phenotype required an interleukin-1 receptor (IL-1R)-dependent mechanism. Indeed, targeting of IL-1-IL-1R axis in a human RCC xenograft model abrogated the protumor phenotype of tumor-associated macrophages (TAMs) and reduced tumor growth in vivo. Supporting this, meta-analysis of gene expression from human RCC tumors showed IL1B expression to correlate with myelomonocytic markers, protumor genes, and tumor staging. Analyzing RCC patient tumors confirmed the protumor phenotype of TAMs. These data provide direct evidence for a tumor-promoting role of monocytes and macrophages in human cancer and indicate IL-1-IL-1R as a possible therapeutic target.

Acute-onset chronic inflammatory demyelinating polyneuropathy with focal segmental glomerulosclerosis.

Inflammatory neuropathies have been reported to occur in association with nephrotic syndrome. Their underlying immuno-pathogenic mechanisms remain unknown. A 50-year-old woman concurrently presented with acute-onset chronic inflammatory demyelinating polyneuropathy and nephrotic syndrome secondary to focal segmental glomerulosclerosis. Both neuropathy and proteinuria improved after plasma exchange and steroids. Literature review of cases of concurrent inflammatory neuropathies and nephrotic syndrome revealed similar neuro-renal presentations. This neuro-renal condition may be mediated by autoantibodies targeting myelin and podocytes.

Carcinoma cuniculatum of the esophagus and tongue: report of two cases, including TP53 mutational analysis.

Carcinoma cuniculatum (CC) is a rare variant of extremely well differentiated squamous cell carcinoma. We present the clinicopathological features of two cases of CC; one lingual and one esophageal case with a molecular genetic study regarding the TP53 gene mutational status. Case 1 was a 62 year old male with enlarging chronic ulcer in the tongue. Case 2 was a 77 year old male with progressive dysphagia and odynophagia. Both patients were treated surgically. Both tumors showed deeply invaginating, keratin-filled, burrowing crypts lined by very well differentiated squamous epithelium. The esophageal tumor showed varying degrees of reactive nuclear atypia largely limited to the areas with dense intratumoral infiltration of neutrophils. No mutation of TP53 was identified in the esophageal case. Cytologic atypia limited to areas of significant acute inflammation may occur in CC and should, in the absence of aggressive stromal invasion, not preclude a diagnosis of CC.

Digital pathology: exploring its applications in diagnostic surgical pathology practice.

There has been a recent upsurge in worldwide attention on digital pathology, which has transformed from static snapshots from camera-equipped microscopes to its modern form that encompasses scanning of whole glass slides with evaluation of histological images on a computer screen, along with management of its accompanying information. Although it has been widely accepted in education and research, its implementation in diagnostic surgical pathology practice is not without challenges in workflow integration, technological infrastructure, pathologist acclimatisation, global standardisation for clinical practice, and cost issues, among others. Nonetheless, early adopters have harnessed its benefits in specific niches, like frozen section services and remote second opinion consultations. Its tremendous potential is worthy of further validation to compare with conventional glass slide evaluation, even while it is already paving the way for advancement into virtual three-dimensional imaging technology, with a glimpse into a possible future digital diagnostic pathology practice.