Insights into MLH1 Methylation in Endometrial Adenocarcinoma through Pyrosequencing Analysis: A Retrospective Observational Study.

Background: In cancer care, the MLH1 gene is crucial for DNA mismatch repair (MMR), serving as a vital tumor suppressor. Evaluating MLH1 protein expression status, followed by analysis of MLH1 promoter methylation, has become a key diagnostic and prognostic approach. Our study investigates the complex link between MLH1 methylation and prognosis in endometrial adenocarcinoma (EA) patients. Methodology: MLH1 methylation status was accessed by a Pyrosequencing (PSQ) assay. Qualitative positivity for methylation was established if it exceeded the 11% cut-off; as well, a quantitative methylation analysis was conducted to establish correlations with clinicopathological data, relapse-free survival, and disease-free survival. Results: Our study revealed that 33.3% of patients without MLH1 methylation experienced relapses, surpassing the 23.3% in patients with methylation. Furthermore, 16.7% of patients without methylation succumbed to death, with a slightly higher rate of 17.6% in methylated patients. Qualitative comparisons highlighted that the mean methylation rate in patients experiencing relapse was 35.8%, whereas in those without relapse, it was 42.2%. This pattern persisted in disease-specific survival (DSS), where deceased patients exhibited a higher mean methylation level of 49.1% compared to living patients with 38.8%. Conclusions: Our findings emphasize the efficacy of PSQ for evaluating MLH1 methylation. While unmethylation appears to be associated with a higher relapse rate, the survival rate does not seem to be influenced by methylation. Quantitative percentages suggest that elevated MLH1 methylation is linked to relapse and mortality, though a study with a larger sample size would be essential for statistically significant results.

Colonization by Extended-Spectrum ß-Lactamase-Producing Enterobacterales and Bacteremia in Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Assessing the risk of multidrug-resistant colonization and infections is pivotal for optimizing empirical therapy in hematopoietic stem cell transplants (HSCTs). Limited data exist on extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) colonization in this population. This study aimed to assess whether ESBL-E colonization constitutes a risk factor for ESBL-E bloodstream infection (BSI) and to evaluate ESBL-E colonization in HSCT recipients. METHODS: A retrospective analysis of ESBL-E colonization and BSI in HSCT patients was conducted from August 2019 to June 2022. Weekly swabs were collected and cultured on chromogenic selective media, with PCR identifying the ß-lactamase genes. Pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) assessed the colonizing strains' similarities. RESULTS: Of 222 evaluated HSCT patients, 59.45% were colonized by ESBL-E, with 48.4% at admission. The predominant ß-lactamase genes were blaTEM (52%) and blaSHV (20%). PFGE analysis did not reveal predominant clusters in 26 E. coli and 15 K. pneumoniae strains. WGS identified ST16 and ST11 as the predominant sequence types among K. pneumoniae. Thirty-three patients developed thirty-five Enterobacterales-BSIs, with nine being third-generation cephalosporin-resistant. No association was found between ESBL-E colonization and ESBL-BSI (p = 0.087). CONCLUSIONS: Although the patients presented a high colonization rate of ESBL-E upon admission, no association between colonization and infection were found. Thus, it seems that ESBL screening is not a useful strategy to assess risk factors and guide therapy for ESBL-BSI in HSCT-patients.

COVID-19 in hematopoietic stem cell transplant recipients during three years of the pandemic: a multicenter study in Brazil.

Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

COVID-19 in hematopoietic stem cell transplant recipients during three years of the pandemic: a multicenter study in Brazil

ABSTRACT Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

Case Report: In Situ and Systemic Immune Response to Mucosal Leishmaniasis in an HIV-Infected Patient.

In situ and systemic evaluations of the immune responses of HIV-infected patients to mucosal leishmaniasis have been poorly described. We describe a recently diagnosed HIV-infected patient with mucosal leishmaniasis who was characterized by a CD4 count of 85 cells/mm3 and nasal septum destruction resulting from pruritic and ulcerated nasal mucosa with crust formation and progression over 2 years. In situ and systemic immune evaluations of T cell activation, memory, and exhaustion were conducted using cytofluorometric assays, and sequencing of the Leishmania species was performed. The immune profile of HIV-infected patient with mucosal leishmaniasis shows a mixed Th1/Th2 pattern and an activated and exhausted status.

Epidemiologic case investigation on the zoonotic transmission of Methicillin-resistant Staphylococcus pseudintermedius among dogs and their owners.

Dogs often carry methicillin-resistant Staphylococci asymptomatically. These bacteria are frequently linked to conditions such as canine pyoderma and otitis. Close interaction between dogs and humans can facilitate the exchange of resistant strains, particularly Methicillin-resistant Staphylococcus pseudintermedius (MRSP). This represents a public health issue, since these strains, in addition to occasionally causing infections in humans, can also serve as a source of resistance and virulence genes for strains of greater importance in human medicine, such as Staphylococcus aureus. Furthermore, MRSP strains are often multidrug resistant, which ends up compromising the treatment of infections. This study aimed to assess the potential transmission of Staphylococcus pseudintermedius among dogs and their owners. We examined a total of one hundred canine samples collected from cases of pyoderma and otitis to detect the presence of staphylococci. Simultaneously, we conducted evaluations on all dog owners. Staphylococci strains were identified using MALDI-TOF MS and PCR targeting the nuc gene. Methicillin resistance screening was also performed by detecting the mecA gene using PCR. Among the sampled dogs, 64 carried S. pseudintermedius. Nine were identified as MRSP. In six instances, dogs and their owners exhibited S. pseudintermedius. These samples underwent genome sequencing and were screened for antimicrobial resistance genes, SCCmec typing, MLST characterization, and Single Nucleotide Polymorphisms (SNP) analyses. The results of the phylogenetic analysis revealed that in three cases, dogs and owners had closely related isolates, suggesting interspecies transmission. Two of these cases involved MRSP and one MSSP. Moreover, in the two MRSP cases, the same SCCmec type (type V) was detected. Additionally, the sequence type was consistent across all three cases involving dogs and owners (MSSP ST2277, MRSP ST2282, and ST2286). These findings strongly indicate a transmission event. Since Staphylococcus pseudintermedius is primarily isolated from canine samples, it is plausible that dogs may have acted as a potential source. In the remaining three cases, despite identifying the same species in both samples, they had notable phylogenetic differences.

Investigation of antimicrobial susceptibility and genetic diversity among Staphylococcus pseudintermedius isolated from dogs in Rio de Janeiro.

Staphylococcus pseudintermedius is an opportunistic pathogen causing a variety of infections that are difficult to treat, especially because of the development of antimicrobial resistance. It has a clonal distribution around the world. To have a better understanding of the MRSP population, we search the presence of MRSP in colonized or infected dogs. Samples from 99 dogs with infections and 35 from asymptomatic dogs were collected. Isolates were identified by mass spectrometry and Multiplex-PCR. The mecA gene was confirmed by conventional PCR. MRSP strains were analyzed by whole-genome sequencing. 75 S. pseudintermedius were identified, most from infection cases. The species were isolated from 70 out of the 135 dogs. Penicillin and Trimethoprim/Sulfamethoxazole presented higher resistance rates. Forty-seven strains were classified as multi-drug resistant (MDR), and were more isolated from dogs with infection (P < 0.05). Eighteen samples were classified as MRSP, representing 24.0% of the population. Six of 16 MRSP sequenced samples belonged to the world spread clone ST71; others belonged to unknown clones. Most samples carried the SCCmec type IIIA. Twenty-one different genetic resistance determinants were found among MRPS strains. MRSP is circulating among infected and colonized dogs in Rio de Janeiro, Brazil.

Encephalopathy Caused by Human Parvovirus B19 Genotype 1 Associated with Haemophilus influenzae Meningitis in a Newborn.

Parvovirus B19 infection is associated with a wide range of clinical manifestations, from asymptomatic to severe neurological disorders. Its major clinical symptoms, fever and rash, are common to multiple viruses, and laboratory tests to detect B19 are frequently not available. Thus, the impact of B19 on public health remains unclear. We report the case of a 38-day old girl admitted to São Paulo Clinical Hospital, Brazil, with an initial diagnosis of bacterial meningitis, seizures, and acute hydrocephalus. Antibiotic therapy was maintained for one week after admission and discontinued after negative laboratory results were obtained. Nine days after symptoms onset, a cerebral spinal fluid (CSF) sample revealed persistent pleocytosis. The complete B19 complete genome was subsequently identified in her CSF by a metagenomic next-generation sequencing approach. This report highlights the possible involvement of B19 in the occurrence of acute neurological manifestations and emphasizes that its possible involvement might be better revealed by the use of metagenomic technology to detect viral agents in clinical situations of unknown or uncertain etiology.

Performance Evaluation of VIDAS® Diagnostic Assays Detecting Anti-Chikungunya Virus IgM and IgG Antibodies: An International Study.

Chikungunya (CHIK) is a debilitating mosquito-borne disease with an epidemiology and early clinical symptoms similar to those of other arboviruses-triggered diseases such as dengue or Zika. Accurate and rapid diagnosis of CHIK virus (CHIKV) infection is therefore challenging. This international study evaluated the performance of the automated VIDAS® anti-CHIKV IgM and IgG assays compared to that of manual competitor IgM and IgG ELISA for the detection of anti-CHIKV IgM and IgG antibodies in 660 patients with suspected CHIKV infection. Positive and negative agreements of the VIDAS® CHIKV assays with ELISA ranged from 97.5% to 100.0%. The sensitivity of the VIDAS® CHIKV assays evaluated in patients with a proven CHIKV infection confirmed reported kinetics of anti-CHIKV IgM and IgG response, with a positive detection of 88.2-100.0% for IgM ≥ 5 days post symptom onset and of 100.0% for IgG ≥ 11 days post symptom onset. Our study also demonstrated the superiority of ELISA and VIDAS® assays over rapid diagnostic IgM/IgG tests. The analytical performance of VIDAS® anti-CHIKV IgM and IgG assays was excellent, with a high precision (coefficients of variation ≤ 7.4%) and high specificity (cross-reactivity rate ≤ 2.9%). This study demonstrates the suitability of the automated VIDAS® anti-CHIKV IgM and IgG assays to diagnose CHIKV infections and supports its applicability for epidemiological surveillance and differential diagnosis in regions endemic for CHIKV.

Chlorhexidine susceptibility and Eagle effect in planktonic cells and biofilm of nosocomial isolates.

The aim of this study is to evaluate the chlorhexidine gluconate (CHG) susceptibility in both planktonic cells and biofilm of 32 Gram-negative (Gn) and 6 Gram-positive (Gp) isolates by minimal inhibitory concentration (2-256 µg/mL for Gn and 2-32 µg/mL for Gp), minimal bactericidal concentration (4-256 µg/mL for Gn and 2-32 µg/mL for Gp) in planktonic cells, and minimal biofilm elimination concentration (128 ≥ 16,384 µg/mL in Gn and 32 ≥ 16,384 µg/mL in Gp) in biofilm environment. Our study showed that Gn isolates have higher minimal concentrations than Gp and bacteria in biofilms are more tolerant than planktonic ones. No correlation between MBC or MBEC and biofilm formation was statistically confirmed. The Eagle effect, previously described for antimicrobials and antifungals, was evidenced in this work for CHG, an antiseptic. Besides that, the phenomenon was described in 23/38 isolates (60.5%), raising minimal concentration up to ≥ 16,384 µg/mL. Our study showed that clinical isolates have a high ability to form biofilm allowing them to tolerate CHG concentrations as high as the ones used in clinical practice. Therefore, attention should be given to the occurrence of this phenomenon to avoid false susceptibility results.

Clinical markers of post-Chikungunya chronic inflammatory joint disease: A Brazilian cohort.

BACKGROUND: Chikungunya-fever (CHIKF) remains a public health major issue. It is clinically divided into three phases: acute, post-acute and chronic. Chronic cases correspond to 25-40% individuals and, though most of them are characterized by long-lasting arthralgia alone, many of them exhibit persistent or recurrent inflammatory signs that define post-Chikungunya chronic inflammatory joint disease (pCHIKV-CIJD). We aimed to identify early clinical markers of evolution to pCHIKV-CIJD during acute and post-acute phases. METHODOLOGY/PRINCIPAL FINDINGS: We studied a prospective cohort of CHIKF-confirmed volunteers with longitudinal clinical data collection from symptoms onset up to 90 days, including a 21-day visit (D21). Of 169 patients with CHIKF, 86 (50.9%) completed the follow-up, from whom 39 met clinical criteria for pCHIKV-CIJD (45.3%). The relative risk of chronification was higher in women compared to men (RR = 1.52; 95% CI = 1.15-1.99; FDR = 0.03). None of the symptoms or signs presented at D0 behaved as an early predictor of pCHIKV-CIJD, while being symptomatic at D21 was a risk factor for chronification (RR = 1.31; 95% CI = 1.09-1.55; FDR = 0.03). Significance was also observed for joint pain (RR = 1.35; 95% CI = 1.12-1.61; FDR = 0.02), reported edema (RR = 3.61; 95% CI = 1.44-9.06; FDR = 0.03), reported hand and/or feet small joints edema (RR = 4.22; 95% CI = 1.51-11.78; FDR = 0.02), and peri-articular edema observed during physical examination (RR = 2.89; 95% CI = 1.58-5.28; FDR = 0.002). Furthermore, patients with no findings in physical examination at D21 were at lower risk of chronic evolution (RR = 0.41, 95% CI = 0.24-0.70, FDR = 0.01). Twenty-nine pCHIKV-CIJD patients had abnormal articular ultrasonography (90.6% of the examined). The most common findings were synovitis (65.5%) and joint effusion (58.6%). CONCLUSION: This cohort has provided important insights into the prognostic evaluation of CHIKF. Symptomatic sub-acute disease is a relevant predictor of evolution to chronic arthritis with synovitis, drawing attention to joint pain, edema, multiple articular involvement including small hand and feet joints as risk factors for chronification beyond three months, especially in women. Future studies are needed to accomplish the identification of accurate and early biomarkers of poor clinical prognosis, which would allow better understanding of the disease's evolution and improve patients' management, modifying CHIKF burden on global public health.

Immunohistological study of the density and distribution of human penile neural tissue: gradient hypothesis.

Immunohistological patterns of density and distribution of neural tissue in the human penis, including the prepuce, are not fully characterized, and effects of circumcision (partial or total removal of the penile prepuce) on penile sexual sensation are controversial. This study analyzed extra- and intracavernosal innervation patterns on the main penile axes using formalin-fixed, paraffin-embedded human adult and fetal penile tissues, single- and double-staining immunohistochemistry and a variety of neural and non-neural markers, with a special emphasis on the prepuce and potential sexual effects of circumcision. Immunohistochemical profiles of neural structures were determined and the most detailed immunohistological characterizations to date of preputial nerve supply are provided. The penile prepuce has a highly organized, dense, afferent innervation pattern that is manifest early in fetal development. Autonomically, it receives noradrenergic sympathetic and nitrergic parasympathetic innervation. Cholinergic nerves are also present. We observed cutaneous and subcutaneous neural density distribution biases across our specimens towards the ventral prepuce, including a region corresponding in the adult anatomical position (penis erect) to the distal third of the ventral penile aspect. We also describe a concept of innervation gradients across the longitudinal and transverse penile axes. Results are discussed in relation to the specialized literature. An argument is made that neuroanatomic substrates underlying unusual permanent penile sensory disturbances post-circumcision are related to heightened neural levels in the distal third of the ventral penile aspect, which could potentially be compromised by deep incisions during circumcision.

Neisseria gonorrhoeae arthritis in a patient with systemic lupus: resistance and virulence profiles.

In this study, we describe a case report of gonococcal arthritis in a Systemic Lupus Erythematosus patient. Although several mechanisms favor disseminated gonococcal infection (DGI) in patients immunosuppressed by SLE, this association is rarely reported in literature. We performed whole genome sequencing (WGS) of the etiologic agent involved and molecular analysis using a global collection of Neisseria gonorrhoeae strains. Ours is the only sample derived from synovial fluid identified in this collection, the others being from the usual anatomical sites. Antimicrobial susceptibility was determined by disk diffusion and Etest, and WGS was conducted to determine multilocus sequence typing profiles, group isolates based on core genome single nucleotide polymorphisms (SNP), and identify virulence genes and antimicrobial resistance determinants. The N. gonorrhoeae samples in the global collection were highly heterogeneous. The SNP tree had a total 19,532 SNPs in 320 samples. Our sample displayed resistance to ciprofloxacin (MIC = 2 µg/mL) and tetracycline (zone diameter = 0 mm) belonged to ST 1588 and was not closely related to any isolate in the global collection of N. gonorrhoeae strains. The isolate had genetic features related to beta-lactam, tetracycline and quinolone resistance. Seventy-one virulence genes were identified in our sample, belonging to the following classes: adherence, efflux pump, immune modulator, invasion, iron uptake, protease and stress adaptation. Moreover, no virulence genes for immune evasion and toxin were identified.

Virulomic Analysis of Multidrug-Resistant Klebsiella pneumoniae Isolates and Experimental Virulence Model Using Danio rerio (Zebrafish).

This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored blaKPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types.

IgG4-related disease diagnosed in a paratesticular pseudotumor simulating malignancy, in a patient with retroperitoneal fibrosis.

IgG4 related disease is a term used to describe a fibroinflammatory condition characterized by storiform fibrosis, inflammation with a dense lymphoplasmocytic infiltrate rich in plasma cells expressing IgG4, and often, if not always, raised serum levels of IgG4. We report a case of a patient with a past history of retroperitoneal fibrosis presenting with a swollen left testicle, who underwent an orchidectomy due to suspicion of malignancy. The surgical specimen revealed a paratesticular pseudotumor with histopathological and immunohistochemical characteristics of IgG4 related disease. To the best of our knowledge, just nine such cases have previously been reported, of which only three were manifestations of systemic disease, as in the present case. Whilst it is important to recognize the clinical and radiological features of this entity, histopathology is often essential in order to confirm the diagnosis.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Shedding and Predictors of Viral Culture Positivity on Vaccinated Healthcare Workers With Mild Coronavirus Disease 2019.

In this prospective cohort of 30 vaccinated healthcare workers with mild Omicron variant infection, we evaluated viral culture, rapid antigen test (RAT), and real-time reverse-transcription polymerase chain reaction (RT-PCR) of respiratory samples at days 5, 7, 10, and 14. Viral culture was positive in 46% (11/24) and 20% (6/30) of samples at days 5 and 7, respectively. RAT and RT-PCR (Ct ≤35) showed 100% negative predictive value (NPV), with positive predictive values (PPVs) of 32% and 17%, respectively, for predicting viral culture positivity. A lower RT-PCR threshold (Ct ≤24) improved culture prediction (PPV = 39%; NPV = 100%). Vaccinated persons with mild Omicron infection are potentially transmissible up to day 7. RAT and RT-PCR might be useful tools for shortening the isolation period.

Selection and Identification of a DNA Aptamer for Multidrug-Resistant Acinetobacter baumannii Using an In-House Cell-SELEX Methodology.

Infections caused by multidrug-resistant A. baumannii are a worldwide health concern with high mortality rates. Rapid identification of this infectious agent is critical as it can easily spread with difficult or no options for treatment. In this context, the development of reliable and economically viable detection and therapeutic methodologies are still challenging. One of the promising solutions is the development of nucleic acid aptamers capable of interacting with bacteria. These aptamers can be used for specific recognition of infectious agents as well as for blocking their functions. Cell-SELEX technology currently allows the selection and identification of aptamers and is flexible enough to target molecules present in an entire bacterial cell without their prior knowledge. However, the aptamer technology is still facing many challenges, such as the complexity of the screening process. Here, we describe the selection and identification of a new aptamer A01, using an in-house whole-cell SELEX-based methodology, against multi-resistant Acinetobacter baumannii, with rapid execution and low cost. In addition, this protocol allowed the identification of the aptamer A01 with the whole A. baumannii cell as a target. The aptamer A01 demonstrated a binding preference to A. baumannii when compared to K. pneumoniae, C. albicans, and S. aureus in fluorescence assays. Although the time-kill assay did not show an effect on bacterial growth, the potential bactericidal or bacteriostatic cannot be totally discarded. The new categorized aptamer (A01) displayed a significant binding affinity to MDR A. baumannii.

Immunohistochemistry subtyping of urothelial carcinoma is feasible in the daily practice.

The preferred treatment of choice in muscle-invasive bladder cancer (MIBC) is usually transurethral resection followed by cystectomy, with neoadjuvant chemotherapy being a second option. As the treatment is associated with relevant side effects, a great effort is being made to improve the selection of patients, with molecular subtyping being one of the main strategies. Our aim was to develop an immunohistochemical algorithm for subtyping MIBCs. After a literature review, we have developed a simple algorithm to subtype MIBCs based on their morphology and three common antibodies: GATA3, CK5/6, and p16. We applied it to 113 muscle-invasive carcinomas. The positivity threshold for GATA3 and CK5/6 was 20% with at least moderate intensity, while p16 was 70% with moderate to intense nuclear and cytoplasmic staining. Cases GATA3 + CK5/6 - were considered luminal, while cases GATA3 - CK5/6 + were classified as nonluminal/basal squamous. Luminal p16 + cases were labeled as genomically unstable and luminal p16 - as Uro-like. Cases GATA3 + CK5/6 + with a predominantly basal pattern were labeled luminal, while diffuse cases were labeled nonluminal/basal squamous. All GATA3-CK5/6 - cases were considered nonluminal and were divided into mesenchymal-like or neuroendocrine, depending on the morphology. We were able to classify the 113 cases as: 82 (72.57%) were luminal, being 47 Uro-like (41.59%) and 35 (30.97%) genomically unstable; 31 (27.43%) were nonluminal, being 24 basal/squamous (21.24%), two (1.76%) mesenchymal-like, and five (4.42%) neuroendocrine like. We have achieved a feasible and cost-effective algorithm to subtype MIBCs from morphological features and the use of three common antibodies. Further studies in external cohorts are necessary to validate these results.

WISP-2 modulates the induction of inflammatory mediators and cartilage catabolism in chondrocytes.

Wnt-1 inducible signaling pathway protein 2 (WISP-2/CCN5) is a recently identified adipokine that has been described as an important mediator of canonical Wnt activation in adipogenic precursor cells. In osteoarthritis (OA), the most common form of arthritis, chondrocytes exhibit aberrant and increased production of pro-inflammatory mediators and matrix degrading enzymes such as IL-1ß and MMP-13. Although recent evidence suggests a role for Wnt signaling in OA physiopathology, little is known about the involvement of WISP-2 in cartilage degradation. In the present study, we determined the expression of WISP-2 in healthy and OA human chondrocytes. WISP-2 expression is modulated along chondrocyte differentiation and downregulated at the onset of hypertrophy by inflammatory mediators. We also investigated the effect of WISP-2 on cartilage catabolism and performed WISP-2 loss-of-function experiments using RNA interference technology in human T/C-28a2 immortalized chondrocytes. We demonstrated that recombinant human WISP-2 protein reduced IL-1ß-mediated chondrocyte catabolism, that IL-1ß and WNT/b-catenin signaling pathways are involved in rhWISP-2 protein and IL-1ß effects in human chondrocytes, and that WISP-2 has a regulatory role in attenuating the catabolic effects of IL-1ß in chondrocytes. Gene silencing of WISP-2 increased the induction of the catabolic markers MMP-13 and ADAMTS-5 and the inflammatory mediators IL-6 and IL-8 triggered by IL-1ß in human primary OA chondrocytes in a Wnt/ß-catenin dependent manner. In conclusion, here we have shown for the first time that WISP-2 may have relevant roles in modulating the turnover of extracellular matrix in the cartilage and that its downregulation may detrimentally alter the inflammatory environment in OA cartilage. We also proved the participation of Wnt/ß-catenin signaling pathway in these processes. Thus, targeting WISP-2 might represent a potential therapeutical approach for degenerative and/or inflammatory diseases of musculoskeletal system, such as osteoarthritis.