Immunohistochemical findings in the lungs of COVID-19 subjects: evidence of surfactant dysregulation.

OBJECTIVE: Acute respiratory distress syndrome (ARDS) is characterized by quantitative and qualitative changes in surfactant composition, leading to surfactant dysregulation with alveolar collapse and acute respiratory hypoxic failure. Recently, surfactant has been hypothesized to play a relevant role in COVID-19, representing a strong defender against SARS-CoV-2 infection. The aim of our work was the study of immunohistochemical surfactant expression in the lungs of patients died following SARS-CoV-2 ARDS, in order to shed light on a possible therapeutic surfactant administration. PATIENTS AND METHODS: We investigated four patients who died due to ARDS following SARS-COV-2 infection and four patients submitted to lung biopsy, in the absence of SARS-CoV-2 infection. In all 8 cases, lung specimens were immunostained with anti-surfactant protein A (SP-A) and B (SP-B). RESULTS: In control subjects, reactivity for SP-B was restricted to type II alveolar cells. Immunostaining for SP-A was observed on the surface of alveolar spaces. In the COVID-19 positive lungs, immunoreactivity for SP-B was similar to that observed in control lungs; SP-A was strongly expressed along the alveolar wall. Moreover, dense aggregates of SP-A positive material were observed in the alveolar spaces. CONCLUSIONS: Our immunohistochemical data show the dysregulation of surfactant production in COVID-19 patients, particularly regarding SP-A expression. The increased presence of SP-A in condensed masses inside alveolar spaces could invalidate the therapeutic efficacy of the treatment with exogenous surfactant.

Differential levels of surfactant protein A, surfactant protein D, and progesterone to estradiol ratio in maternal serum before and after the onset of severe early-onset preeclampsia.

PROBLEM: Preeclampsia (PE), a multifactorial disorder characterized by impaired placental development, elevated inflammatory response and dysregulated placental steroidogenesis. PE may be preventable if predicted early on. METHOD OF STUDY: The study evaluated the potential of immunomodulatory collectins, surfactant protein A (SP-A), surfactant protein D (SP-D), and mannose binding lectin (MBL), to predict PE before the disease onset, in a prospective study cohort of healthy pregnant women (n = 922). In addition, a cross-sectional study was conducted to determine the serum and placental profile of collectins in PE women after the disease onset (early-onset PE [EOPE], n = 33; late-onset PE [LOPE], n = 24); and controls [n = 75]. The serum profiles of estradiol (E2) and progesterone (P4) were evaluated to determine their correlation with collectins. RESULTS: In the prospective cohort, significantly decreased serum levels of SP-A, SP-D, P4/E2 ratio were observed in women who subsequently developed severe EOPE. Interestingly, after the disease onset, there was a significant increase in serum and placental levels of collectins in women with severe EOPE, whereas women with LOPE had significantly decreased levels of collectins. Serum P4/E2 ratio was significantly altered in severe EOPE and positively correlated with serum levels of SP-A and SP-D. CONCLUSION: Collectins are differentially expressed in the serum during progression of PE. Decreased serum levels of SP-A, SP-D, P4/E2 ratio and increased E2 during 10-20 weeks of gestation are novel plausible risk factors for early prediction of EOPE in Indian women.

SP-A and TLR4 localization in lung tissue of SM-exposed patients.

INTRODUCTION: Long-term pulmonary complications are one of the major long-term consequences of sulfur mustard (SM) exposure. Toll-like receptor 4 (TLR4) involves in the pathogenesis of several pulmonary disorders. Surfactant protein-A (SP-A) regulates LPS-induced TLR4 localization and activation responses. However, the intensity and significance of TLR4 and SP-A expression by lung cells in SM-exposed patients is not clear. METHODS: The gene expression of TLR4 (through real-time PCR) and TLR4 and SP-A positive cells and alveolar type II cells, as SP-A producers, (using IHC) were assessed in formalin fixed paraffin embedded (FFPE) specimens from SM-exposed (n = 17), and non-SM exposed individuals (n = 12). RESULTS: TLR4 gene expression did not change between study groups. However, its cell surface presentation was significantly reduced in SM-exposed patients and particularly in which with constrictive bronchiolitis compared with the control group (P < 0.001 and P = 0.002, respectively). Frequency of alveolar type II cells was lower in the case group rather than the control group while the number of SP-A positive cells did not alter. CONCLUSIONS: These findings suggest that reduced TLR4 cell surface presentation may have anti-inflammatory function and SP-A may have a critical role in regulation of inflammatory responses in SM-exposed patients. Further investigation on other possible mechanisms involved in TLR4 internalization maybe help to illustrate the modulatory or inflammatory activity of TLR4 in these patients.

Change of surfactant protein D and A after renal ischemia reperfusion injury.

Acute kidney injury (AKI) is associated with widespread effects on distant organs, including the lungs. Surfactant protein (SP)-A and SP-D are members of the C-type lectin family, which plays a critical role in host defense and regulation of inflammation in a variety of infections. Serum levels of SP-A and SP-D are markers to reflect lung injury in acute respiratory distress syndrome, idiopathic pulmonary fibrosis, and sarcoidosis. We investigated the change of lung-specific markers, including SP-A and SP-D in an AKI mice model. We studied C57BL/6J mice 4 and 24 hours after an episode of ischemic AKI (23 min of renal pedicle clamping and then reperfusion); numerous derangements were present, including SP-A, SP-D, and lung tight-junction protein. Neutrophil infiltration and apoptosis in the lungs increased in ischemic AKI. Receptor for advanced glycation end products (RAGE) in the lungs, a marker of pneumocyte I, was not changed. Lung tight-junction proteins, particularly claudin-4, claudin-18, and anti-junctional adhesion molecule 1 (JAMA-1), were reduced in 24 hours after AKI. Serum SP-A and SP-D significantly increased in ischemic AKI. SP-A and SP-D in the lungs did not increase in ischemic AKI. The immunohistochemistry showed that the expression of SP-A and SP-D was intact in ischemic AKI. SP-A and SP-D in the kidneys were significantly higher in AKI than in the sham. These patterns of SP-A and SP-D in the kidneys were similar to those of serum. AKI induces apoptosis and inflammation in the lungs. Serum SP-A and SP-D increased in ischemic AKI, but these could have originated from the kidneys. So serum SP-A and SP-D could not reflect lung injury in AKI. Further study is needed to reveal how a change in lung tight-junction protein could influence the prognosis in patients with AKI.

Surfactant Protein A in particles in exhaled air (PExA), bronchial lavage and bronchial wash - a methodological comparison.

INTRODUCTION: At present, there are few methods available for monitoring respiratory diseases affecting distal airways. Bronchoscopy is the golden standard for sampling the lower airways. The recently developed method for collecting non-volatile material from exhaled air - PExA (Particles in Exhaled air) is a promising new tool, but no direct comparison between the two methods has yet been performed. The aim of the present study was to compare sampling using PExA with bronchial wash (BW) representing the larger more proximal airways and broncho-alveolar lavage (BAL) representing the distal airways. METHODS: 15 healthy non-smoking subjects (7 female/8 male), age 28 ± 4 years, with normal lung function were included in the study. PExA-sampling (2 × 250 ng particles) and bronchoscopy with BW (2 × 20 ml) and BAL (3 × 60 ml sterile saline) was performed. Albumin and Surfactant Protein A (SP-A) were analyzed with ELISA, and analyses of correlation were performed. RESULTS: A significant association was found between BAL-fluid albumin and PExA-albumin (rs:0.65 p = 0.01). There was also an association between SP-A in PExA and BAL, when corrected for albumin concentration (rs:0.61, p = 0.015). When correlating concentrations of albumin and SP-A in bronchial wash and PExA respectively, no associations were found. CONCLUSIONS: This is the first direct comparison between the bronchoscopy-based BW/BAL-fluids and material collected using the PExA methodology. Both albumin and albumin-corrected SP-A concentrations were significantly associated between BAL and PExA, however, no such association was found in either marker between BW and PExA. These results indicate that the PExA method samples the distal airways. PExA is thus considered a new promising non-invasive assessment for monitoring of the distal airways.

Immunohistochemical expression of P-selectin, SP-A, HSP70, aquaporin 5, and fibronectin in saltwater drowning and freshwater drowning.

The diagnosis of drowning is one of the most difficult in forensic medicine. The aim of this study was to analyze pulmonary tissue reactions in death by drowning. In particular, we focused on the immunohistochemical expression of P-selectin, SP-A, HSP70, AQP-5, and fibronectin to investigate our expression in drowning and to understand whether there are differences between saltwater drowning (SWD) and freshwater drowning (FWD), which may indicate a different pathophysiology. We retrospectively investigated 10 cases of SWD (Mediterranean Sea) from the Institute of Legal Medicine of Genoa (Italy), and 10 cases of FWD (Lake of Geneva) from the University Center of Legal Medicine of Geneva (Switzerland). As control group, we examined 10 cases of death by acute external bleeding, characterized by minimal respiratory distress. As compared with controls, in SWD cases, the results showed a decrease of SP-A expression with membrane patterns. Furthermore, we observed a greater SP-A expression with granular pattern in drowning cases without statistically significant difference between SWD and FWD. For the markers AQP-5, HSP70, fibronectin, and P-selectin, no statistically significant differences were found between SWD, FWD, and controls.

Pirfenidone Improves Familial Idiopathic Pulmonary Fibrosis without Affecting Serum Periostin Levels.

Background: Antifibrotic agents have been approved for the treatment of idiopathic pulmonary fibrosis (IPF). However, the efficacy of these drugs in the treatment of familial IPF (FIPF) has not been previously reported. Case presentation: We report the case of a 77-year-old man with FIPF, successfully treated with pirfenidone. His uncle died due to IPF, and his niece was diagnosed with the disease. He had worsening dyspnea two months prior to admission to our hospital. Upon admission, he had desaturation when exercising and broad interstitial pneumonia. Administration of pirfenidone improved his dyspnea, desaturation, and the reticular shadow on his chest radiograph. Increased fibrotic marker levels KL-6 and SP-D were also normalized in six months; treatment had no effect on his serum periostin level. Pirfenidone has been effective for over two years. Conclusion: Antifibrotic agents such as pirfenidone may be useful for the management of FIPF, as well as cases of sporadic IPF.

Significance of molecular biomarkers in idiopathic pulmonary fibrosis: A mini review.

Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible condition with poor prognosis that is characterized by a variable clinical course in each patient, which renders it a complex disease with unknown causes. Despite the proven efficacy of novel antifibrotic therapies, including pirfenidone and nintedanib, the diagnosis and follow-up of IPF remain challenging. Hence, the identification of molecular biomarkers for early detection of IPF and to predict biologically determined individual clinical courses, has recently piqued the interest of researchers. Previous studies have demonstrated the diagnostic and prognostic efficacy of blood proteins such as KL-6, Surfactant protein (SP)-A, and SP-D, in patients with IPF. Due to their use in clinical practice in Japan, for approximately twenty years, a significant amount of data about these biomarkers has been accumulated. This paper reviews the recent literature on molecular biomarkers for IPF that have been developed in Japan as well as other potential molecular biomarkers.

[Clinical value of surfactant protein-A in exudate pleural effusion].

OBJECTIVE: To evaluate the clinical value of surfactant protein-A (SP-A) in exudate pleural effusion (EPE).
 Methods: This clinical study was prospective, observational and cross-sectional. Two hundred and fifteen patients with pleural effusion were divided into the transudate pleural effusions (TPE) group and the EPE group. TPE patients served as the control group. The concentrations of pleural effusions SP-A (SP-Apl) and serum SP-A (SP-Ase) were measured by ELISA, and receiver operator characteristic (ROC) curve and multivarate Cox analysis of SP-A was analysed for its clinical value.
 Results: SP-Apl concentrations in the EPE group were significantly higher than that in the TPE group [(189.8±43.4) ng/mL vs (22.3±5.1) ng/mL, P<0.01]; SP-Ase concentrations in the EPE group were higher than that in the TPE group [(78.9±11.3) ng/mL vs (25.8±12.4) ng/mL, P<0.05]; SP-Apl concentrations were significantly higher than the concentrations of SP-Ase in the EPE group (P<0.01). In EPE group, SP-Apl and SP-Ase concentration in the patients with primary lung adenocarcinomas were the highest. The cut off value of SP-Apl concentrations was more than 484.5 ng/mL, yielding a 85.4% sensitivity and 95.2% specificity for diagnosing primary lung adenocarcinomas, with an area under the curve (AUC) of 0.943 (95% CI 0.852 to 0.934, P<0.01); when SP-Ase concentration was more than 84.2 ng/mL, it yielded a 76.4% sensitivity and 94.3% specificity for diagnosing primary lung adenocarcinomas, with an AUC of 0.910 (95% CI 0.921 to 0.953, P<0.01).
 Conclusion: While SP-Apl concentration is more than 484.5 ng/mL and/or SP-Ase concentration is more than 84.2 ng/mL, it may be helpful for the diagnosis of primary lung adenocarcinomas with the usage of pleural effusion.

Immunolocalization of Surfactant Proteins SP-A, SP-B, SP-C, and SP-D in Infantile Labial Glands and Mucosa.

Surfactant proteins in different glandular structures of the oral cavity display antimicrobial activity for protection of invading microorganisms. Moreover, they are involved in lowering liquid tension in fluids and facilitate secretion flows. Numerous investigations for studying the occurrence of surfactant proteins in glandular tissues were performed using different methods. In the oral cavity, minor salivary glands secrete saliva continuously for the maintenance of a healthy oral environment. For the first time, we could show that infantile labial glands show expression of the surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D in acinar cells and the duct system in different intensities. The stratified squamous epithelium of the oral mucosa revealed positive staining for SPs in various cell layers.

Arterial Carboxyhemoglobin Measurement Is Useful for Evaluating Pulmonary Inflammation in Subjects with Interstitial Lung Disease.

Objective The arterial concentration of carboxyhemoglobin (CO-Hb) in subjects with inflammatory pulmonary disease is higher than that in healthy individuals. We retrospectively analyzed the relationship between the CO-Hb concentration and established markers of disease severity in subjects with interstitial lung disease (ILD). Methods The CO-Hb concentration was measured in subjects with newly diagnosed or untreated ILD and the relationships between the CO-Hb concentration and the serum biomarker levels, lung function, high-resolution CT (HRCT) findings, and the uptake in gallium-67 (67Ga) scintigraphy were evaluated. Results Eighty-one non-smoking subjects were studied (mean age, 67 years). Among these subjects, (A) 17 had stable idiopathic pulmonary fibrosis (IPF), (B) 9 had an acute exacerbation of IPF, (C) 44 had stable non-IPF, and (D) 11 had an exacerbation of non-IPF. The CO-Hb concentrations of these subjects were (A) 1.5±0.5%, (B) 2.1±0.5%, (C) 1.2±0.4%, and (D) 1.7±0.5%. The CO-Hb concentration was positively correlated with the serum levels of surfactant protein (SP)-A (r=0.38), SP-D (r=0.39), and the inflammation index (calculated from HRCT; r=0.57) and was negatively correlated with the partial pressure of oxygen in the arterial blood (r=-0.56) and the predicted diffusion capacity of carbon monoxide (r=-0.61). The CO-Hb concentrations in subjects with a negative heart sign on 67Ga scintigraphy were higher than those in subjects without a negative heart sign (1.4±0.5% vs. 1.1±0.3%, p=0.018). Conclusion The CO-Hb levels of subjects with ILD were increased, particularly during an exacerbation, and were correlated with the parameters that reflect pulmonary inflammation.

Microbiota Composition and Pulmonary Surfactant Protein Expression as Markers of Death by Drowning.

Pathological diagnosis of drowning remains a challenge for forensic science, because of a lack of pathognomonic findings. We analyzed microbiota and surfactant protein in the lungs for a novel diagnosis of drowning. All rats were divided into drowning, postmortem submersion, and control groups. The water, lungs, closed organs (kidney and liver), and cardiac blood in rats were assayed by targeting 16S ribosomal RNA of Miseq sequencing. Lung samples were analyzed by immunohistochemical staining for surfactant protein A. The closed organs and cardiac blood of drowned group have a lot of aquatic microbes, which have not been detected in postmortem submersion group. Furthermore, intra-alveolar granular staining of surfactant protein A (SP-A) was severely observed in the drowned group than the postmortem submersion and control groups. The findings suggested that the presence of aquatic microbiota in the closed organs and increased expression of SP-A could be markers for a diagnosis of drowning.

Values of SP-A protein in the nasal mucosa.

The paranasal sinus epithelium is exposed to the environment and therefore to a variety of biological, chemical and mechanical insults. Surfactant protein A (SP-A) is a 34-36 kD pulmonary surfactant-associated protein that appears to play an important role in mammalian first-line host defence. Recent studies have reported the possibility of local production of SP-A in the extrapulmonary organs and tissues of the human body. However, the presence of SP-A in the human paranasal sinus mucosa is not well known. The purpose of this study was to investigate the expression of SP-A protein in human turbinate mucosa and to compare the expression of SP-A mRNA in normal turbinate mucosa and turbinate mucosa of chronic rhinosinusitis patients. Reverse transcriptase polymerase chain reaction was used to detect SP-A mRNA. Student's t test was used for statistical comparison of the SP-A/GAPDH-mRNA ratio (GAPDH: glycerinaldehyde-3-phosphate dehydrogenase) of cases and controls. We found expression of SP-A mRNA in mucosa lining the inferior turbinates of healthy patients and its up-regulation in mucosa lining the inferior turbinates of patients with chronic rhinosinusitis. These results may provide targets for new therapies for chronic rhinosinusitis.

[Levels of surfactant proteins A and D in bronchoalveolar lavage fluid of children with pneumonia and their relationships with clinical characteristics].

OBJECTIVE: To observe the levels of pulmonary surfactant proteins A and D (SP-A, SP-D) in bronchoalveolar lavage fluid (BALF) of children with pneumonia, and to explore their relationships with clinical characteristics. METHODS: Thirty-five children with pneumonia were enrolled in this study. Differential cell counts were obtained by Countstar counting board. The levels of SP-A and SP-D in BALF were detected using ELISA. RESULTS: In children with pneumonia, SP-D levels were significantly higher than SP-A levels (P<0.001). SP-D levels were negatively correlated with the neutrophil percentage in BALF (r(s)=-0.5255, P<0.01). SP-D levels in BALF in children with increased blood C-reactive protein levels (>8 mg/L) were significantly lower than in those with a normal level of C-reactive protein (P<0.05). Compared with those in children without wheezing, SP-D levels in children with wheezing were significantly lower (P<0.01). There was no correlation between SP-A levels and clinical characteristics. CONCLUSIONS: SP-D levels in BALF are significantly higher than SP-A levels, and have a certain correlation with clinical characteristics in children with pneumonia. As a protective factor, SP-D plays a more important role than SP-A in regulating the immune and inflammatory responses.

IgA modulates respiratory dysfunction as a sequela to pulmonary chlamydial infection as neonates.

Neonatal Chlamydia lung infections are associated with serious sequelae such as asthma and airway hyper-reactivity in children and adults. Our previous studies demonstrated the importance of Th-1 type cytokines, IL-12 and IFN-γ in protection against neonatal pulmonary chlamydial challenge; however, the role of the humoral arm of defense has not been elucidated. We hypothesized that B-cells and IgA, the major mucosal antibody, play a protective role in newborns against development of later life respiratory sequelae to Chlamydia infection. Our studies using neonatal mice revealed that all WT and IgA-deficient (IgA(-/-)) animals survived a sublethal pulmonary Chlamydia muridarum challenge at one day after birth with similar reduction in bacterial burdens over time. In contrast, all B-cell-deficient (µMT) mice succumbed to infection at the same challenge dose correlating to failure to control bacterial burdens in the lungs. Although IgA may not be important for bacterial clearance, we observed IgA(-/-) mice displayed greater respiratory dysfunction 5 weeks post challenge. Specifically, comparative respiratory functional analyses revealed a significant shift upward in P-V loops, and higher dynamic resistance in IgA(-/-) animals. This study provides insight(s) into the protective role of IgA in neonates against pulmonary chlamydial infection induced respiratory pathological sequelae observed later in life.

Distinct compartmentalization of SP-A and SP-D in the vasculature and lungs of patients with idiopathic pulmonary fibrosis.

BACKGROUND: Surfactant proteins SP-A and SP-D are useful biomarkers in diagnosis, monitoring, and prognosis of idiopathic pulmonary fibrosis (IPF). Despite their high structural homology, their serum concentrations often vary in IPF patients. This retrospective study aimed to investigate distinct compartmentalization of SP-A and SP-D in the vasculature and lungs by bronchoalveolar lavage fluid (BALF)/serum analysis, hydrophilicity and immunohistochemistry. METHODS: We included 36 IPF patients, 18 sarcoidosis (SAR) patients and 20 healthy subjects. Low-speed centrifugal supernatants of BALF (Sup-1) were obtained from each subject. Sera were also collected from each patient. Furthermore, we separated Sup-1 of IPF patients into hydrophilic supernatant (Sup-2) and hydrophobic precipitate (Ppt) by high-speed centrifugation. We measured SP-A and SP-D levels of each sample with the sandwich ELISA technique. We analyzed the change of the BALF/serum level ratios of the two proteins in IPF patients and their hydrophilicity in BALF. The distribution in the IPF lungs was also examined by immunohistochemical staining. RESULTS: In BALF, SP-A levels were comparable between the groups; however, SP-D levels were significantly lower in IPF patients than in others. Although IPF reduced the BALF/serum level ratios of the two proteins, the change in concentration of SP-D was more evident than SP-A. This suggests a higher disease impact for SP-D. Regarding hydrophilicity, although more than half of the SP-D remained in hydrophilic fractions (Sup-2), almost all of the SP-A sedimented in the Ppt with phospholipids. Hydrophilicity suggests that SP-D migrates into the blood more easily than SP-A in IPF lungs. Immunohistochemistry revealed that SP-A was confined to thick mucus-filling alveolar space, whereas SP-D was often intravascular. This data also suggests that SP-D easily leaks into the bloodstream, whereas SP-A remains bound to surfactant lipids in the alveolar space. CONCLUSIONS: The current study investigated distinct compartmentalization of SP-A and SP-D in the vasculature and lungs. Our results suggest that serum levels of SP-D could reflect pathological changes of the IPF lungs more incisively than those of SP-A.

Surfactant protein A is decreased in the lung of rats with hepatopulmonary syndrome.

PURPOSE: To evaluate surfactant protein A levels in an hepatopulmonary syndrome rat model. To date, there have been no studies aimed at evaluating surfactant levels in the setting of cirrhosis or hepatopulmonary syndrome. METHODS: A total of 35 rats were divided into control, sham, and experimental HPS groups. We evaluated surfactant protein A levels in rats and the experimental model designed to induce hepatopulmonary syndrome was common bile duct ligation. Statistical analysis was performed using GraphPad Prism Software(r). Differences were considered statistically significant when p<0.05. RESULTS: Lung homogenate of surfactant protein A levels were lower in the experimental hepatopulmonary syndrome and sham groups in comparison to the control group (p<0.05). Serum SP-A levels were the same in experimental hepatopulmonary syndrome and control groups but decreased in the sham group compared with the experimental groups (p<0.05). Myeloperoxidase activity was higher in the experimental hepatopulmonary syndrome group than the other two groups (p<0.05). CONCLUSION: Surfactant protein A is present in experimental hepatopulmonary syndrome and leads to an imbalance between serum and pulmonary levels due to systemic inflammatory response.

Surfactant protein A is decreased in the lung of rats with hepatopulmonary syndrome

PURPOSE: To evaluate surfactant protein A levels in an hepatopulmonary syndrome rat model. To date, there have been no studies aimed at evaluating surfactant levels in the setting of cirrhosis or hepatopulmonary syndrome. METHODS: A total of 35 rats were divided into control, sham, and experimental HPS groups. We evaluated surfactant protein A levels in rats and the experimental model designed to induce hepatopulmonary syndrome was common bile duct ligation. Statistical analysis was performed using GraphPad Prism Software(r). Differences were considered statistically significant when p<0.05. RESULTS: Lung homogenate of surfactant protein A levels were lower in the experimental hepatopulmonary syndrome and sham groups in comparison to the control group (p<0.05). Serum SP-A levels were the same in experimental hepatopulmonary syndrome and control groups but decreased in the sham group compared with the experimental groups (p<0.05). Myeloperoxidase activity was higher in the experimental hepatopulmonary syndrome group than the other two groups (p<0.05). CONCLUSION: Surfactant protein A is present in experimental hepatopulmonary syndrome and leads to an imbalance between serum and pulmonary levels due to systemic inflammatory response. .

Markers of mechanical asphyxia: immunohistochemical study on autoptic lung tissues.

Forensic pathologists are often asked to provide evidence of asphyxia death in the trial and a histological marker of asphyxiation would be of great help. Data from the literature indicate that the reaction of lung tissue cells to asphyxia may be of more interest for forensic purposes than migrating cells. The lungs of 62 medico-legal autopsy cases, 34 acute mechanical asphyxia (AMA), and 28 control cases (CC), were immunostained with anti-P-selectin, anti-E-selectin, anti-SP-A, and anti-HIF1-α antibodies, in order to verify if some of them may be used as markers of asphyxia death. Results show that P- and E-selectins expression in lung vessels, being activated by several types of trigger stimuli not specific to hypoxia, cannot be used as indicator of asphyxia. Intra-alveolar granular deposits of SP-A seem to be related to an intense hypoxic stimulus, and when massively present, they can suggest, together with other elements, a severe hypoxia as the mechanism of death. HIF1-α was expressed in small-, medium-, and large-caliber lung vessels of the vast majority of mechanical asphyxia deaths and CO intoxications, with the number and intensity of positive-stained vessels increasing with the duration of the hypoxia. Although further confirmation studies are required, these preliminary data indicate an interesting potential utility of HIF1-α as a screening test for asphyxia deaths.

Genotype-phenotype correlation in Chinese patients with pulmonary mixed type adenocarcinoma: Relationship between histologic subtypes, TITF-1/SP-A expressions and EGFR mutations.

This study aimed to explore the association between adenocarcinoma-related morphological and molecular characteristics and EGFR mutations in Chinese lung adenocarcinomas. A total of 139 consecutively resected pulmonary adenocarcinoma patients were screened for EGFR mutations by the amplification refractory mutation system assay. For the resected specimens, histologic subtypes were sub-classified using either the 2004 WHO classification or the 2011 IASLC/ATS/ERS classification. Meanwhile, TITF-1 (thyroid transcription factor 1) and SP-A (surfactant-associated protein A) immunohistochemistry staining was also detected. The results were correlated with EGFR mutations and clinicopathological features mentioned above. Both sub-classification methods reflected differences in frequencies of EGFR mutations in lung adenocarcinoma subtypes. In summary, mixed non-mucinous bronchioloalveolar carcinoma (BAC) or papillary components and papillary predominant adenocarcinoma showed a higher frequency of EGFR mutations than mucinous BAC components; Also, EGFR mutations were significantly more common in tumors with TITF-1 or SP-A expressions than in those without (p=0.002, p=0.026), especially the sensitivity of TITF-1 (96.9%) and the negative predictive value of TITF-1 (88.2%). Our data further showed significant genotype-phenotype correlations between EGFR mutations and adenocarcinoma-related morphological and molecular characteristics, and patients with special histologic and IHC staining features might have higher EGFR mutation rates. In addition, this study, for the first time, indicated the significant relationship between SPA IHC and EGFR mutations, which needed confirmation by further research.