Hassan Y. Dawood

The Villain Team-Up or how Trichomonas vaginalis and bacterial vaginosis alter innate immunity in concert

Objectives Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia). Methods Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells. Results TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines. Conclusions These molecular host–parasite–endosymbiont–bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.

Urinary Excretion of C5b-9 in Severe Preeclampsia: Tipping the Balance of Complement Activation in Pregnancy

The complement cascade is activated in normal pregnancy, and excessive complement activation propagates the systemic inflammatory response in severe preeclampsia. Consequently, biomarkers of complement dysregulation may be useful for prediction or treatment of disease. Because renal damage with proteinuria is a characteristic pathological feature of preeclampsia, we hypothesized that complement markers in urine, rather than plasma, could better reflect complement dysregulation in disease. To investigate this, we performed a case–control study of pregnant women, enrolling 25 cases with severe preeclampsia, 25 controls with chronic hypertension, and 25 healthy controls without hypertension matched by gestational age and parity. Subjects were recruited from the Brigham and Women’s Hospital from March 2012 to March 2013. Urine and blood samples were collected on the day of enrollment, with complement activation (C3a, C5a, and C5b-9) measured by ELISA. Severe preeclampsia was associated with marked elevations in urinary C5b-9 (median [interquartile range], 4.3 [1.2–15.1] ng/mL) relative to subjects with chronic hypertension (0 [0–0]) and healthy controls (0 [0–0]; P <0.0001). Urinary excretion of C5b-9 was detected in 96% of cases with severe preeclampsia, 12% of controls with chronic hypertension, and 8% of healthy controls. Cases were also notable for significantly greater urinary excretion of C3a and C5a. Plasma levels of C5a and C5b-9, but not C3a, were increased in the cases with severe preeclampsia compared with healthy controls; however, they did not distinguish preeclampsia from chronic hypertension, supporting our hypothesis that complement markers in urine, rather than plasma, better reflect complement dysregulation. Complement inhibition is an intriguing treatment option for patients with severe preeclampsia. # Novelty and Significance {#article-title-54}The complement cascade is activated in normal pregnancy, and excessive complement activation propagates the systemic inflammatory response in severe preeclampsia. Consequently, biomarkers of complement dysregulation may be useful for prediction or treatment of disease. Because renal damage with proteinuria is a characteristic pathological feature of preeclampsia, we hypothesized that complement markers in urine, rather than plasma, could better reflect complement dysregulation in disease. To investigate this, we performed a case–control study of pregnant women, enrolling 25 cases with severe preeclampsia, 25 controls with chronic hypertension, and 25 healthy controls without hypertension matched by gestational age and parity. Subjects were recruited from the Brigham and Women’s Hospital from March 2012 to March 2013. Urine and blood samples were collected on the day of enrollment, with complement activation (C3a, C5a, and C5b-9) measured by ELISA. Severe preeclampsia was associated with marked elevations in urinary C5b-9 (median [interquartile range], 4.3 [1.2–15.1] ng/mL) relative to subjects with chronic hypertension (0 [0–0]) and healthy controls (0 [0–0]; P<0.0001). Urinary excretion of C5b-9 was detected in 96% of cases with severe preeclampsia, 12% of controls with chronic hypertension, and 8% of healthy controls. Cases were also notable for significantly greater urinary excretion of C3a and C5a. Plasma levels of C5a and C5b-9, but not C3a, were increased in the cases with severe preeclampsia compared with healthy controls; however, they did not distinguish preeclampsia from chronic hypertension, supporting our hypothesis that complement markers in urine, rather than plasma, better reflect complement dysregulation. Complement inhibition is an intriguing treatment option for patients with severe preeclampsia.

Bacterial Vaginosis and Subclinical Markers of Genital Tract Inflammation and Mucosal Immunity

Bacterial vaginosis (BV) has been linked to an increased risk of human immunodeficiency virus (HIV) acquisition and transmission in observational studies, but the underlying biological mechanisms are unknown. We measured biomarkers of subclinical vaginal inflammation, endogenous antimicrobial activity, and vaginal flora in women with BV and repeated sampling 1 week and 1 month after completion of metronidazole therapy. We also compared this cohort of women with BV to a healthy control cohort without BV. A longitudinal, open label study of 33 women with a Nugent score of 4 or higher was conducted. All women had genital swabs, cervicovaginal lavage (CVL) fluid, and cervicovaginal biopsies obtained at enrollment and received 7 days of metronidazole treatment. Repeat sampling was performed approximately 1 week and 1 month after completion of therapy. Participants baseline samples were compared to a healthy, racially matched control group (n=13) without BV. The CVL from women with resolved BV (Nugent 0-3) had significantly higher anti-HIV activity, secretory leukocyte protease inhibitor (SLPI), and growth-related oncogene alpha (GRO-α) levels and their ectocervical tissues had significantly more CD8 cells in the epithelium. Women with persistent BV after treatment had significantly higher levels of interleukin-1β, tumor necrosis factor alpha (TNF-α), and intercellular adhesion molecule 1 (ICAM-1) in the CVL. At study entry, participants had significantly greater numbers of CCR5(+) immune cells and a higher CD4/CD8 ratio in ectocervical tissues prior to metronidazole treatment, compared to a racially matched cohort of women with a Nugent score of 0-3. These data indicate that BV is associated with changes in select soluble immune mediators, an increase in HIV target cells, and a reduction in endogenous antimicrobial activity, which may contribute to the increased risk of HIV acquisition.

Clinical Evidence for the Role of Trichomonas vaginalis in Regulation of Secretory Leukocyte Protease Inhibitor in the Female Genital Tract

BACKGROUND Secretory leukocyte protease inhibitor (SLPI) is responsible for regulating inflammatory damage to and innate and adaptive immune responses in the vaginal mucosa. Depressed cervicovaginal SLPI levels have been correlated with both Trichomonas vaginalis infection and poor reproductive health outcomes. METHODS We measured levels of SLPI in 215 vaginal specimens collected from adolescent and young adult females aged 14-22 years. Log-transformed SLPI values were compared by analysis of variance or by an unpaired t test before and after adjustment for confounding effects through the propensity score method. RESULTS Females receiving hormonal contraceptives and those with an abnormal vaginal pH had lower SLPI levels as compared to their peers. After propensity score adjustment for race, behavioral factors, hormonal use, and other sexually transmitted infections (STIs), SLPI levels were lower in females with a positive T. vaginalis antigen test result, a vaginal pH >4.5, vaginal leukocytosis, and recurrent (vs initial) T. vaginalis infection, with the lowest levels observed in those with the highest T. vaginalis loads. CONCLUSIONS The SLPI level was reduced by >50% in a T. vaginalis load-dependent manner. Future research should consider whether identifying and treating females with low levels of T. vaginalis infection (before they become wet mount positive) would prevent the loss of SLPI and impaired vaginal immunity. The SLPI level could be used as a vaginal-health marker to evaluate interventions and vaginal products.

Urinary excretion of C5b-9 is associated with the anti-angiogenic state in severe preeclampsia

Severe preeclampsia has been independently linked to complement dysregulation and angiogenic imbalance; however, the relationship between complement and angiogenic factors in human pregnancy is unclear.

Complement Activation and Kidney Injury Molecule-1–Associated Proximal Tubule Injury in Severe Preeclampsia

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case–control study of pregnant women from Brigham and Women’s Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, &bgr;2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase–associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase–associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity.

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.

Venous thromboembolism and intracranial hemorrhage after craniotomy for primary malignant brain tumors: a National Surgical Quality Improvement Program analysis

Venous thromboembolism (VTE), including deep venous thrombosis (DVT) and pulmonary embolism (PE), frequently complicates the postoperative course of primary malignant brain tumor patients. Thromboprophylactic anticoagulation is commonly used to prevent VTE at the risk of intracranial hemorrhage (ICH). We extracted all patients who underwent craniotomy for a primary malignant brain tumor from the National Surgical Quality Improvement Program (NSQIP) registry (2005–2015) to perform a time-to-event analysis and identify relevant predictors of DVT, PE, and ICH within 30 days after surgery. Among the 7376 identified patients, the complication rates were 2.6, 1.5, and 1.3% for DVT, PE, and ICH, respectively. VTE was the second-most common major complication and third-most common reason for readmission. ICH was the most common reason for reoperation. The increased risk of VTE extends beyond the period of hospitalization, especially for PE, whereas ICH occurred predominantly within the first days after surgery. Older age and higher BMI were overall predictors of VTE. Dependent functional status and longer operative times were predictive for VTE during hospitalization, but not for post-discharge events. Admission two or more days before surgery was predictive for DVT, but not for PE. Preoperative steroid usage and male gender were predictive for post-discharge DVT and PE, respectively. ICH was associated with various comorbidities and longer operative times. This multicenter study demonstrates distinct critical time periods for the development of thrombotic and hemorrhagic events after craniotomy. Furthermore, the VTE risk profile depends on the type of VTE (DVT vs. PE) and clinical setting (hospitalized vs. post-discharge patients).

Predicting Readmission and Reoperation for Benign Cranial Nerve Neoplasms: a Nationwide Analysis

OBJECTIVE Readmission and reoperation are risks in the resection of benign cranial nerve tumors (BCNTs). This report analyzes the impact of patient-level and surgical factors on these adverse outcomes. METHODS This retrospective cohort review comprised patients with a diagnosis of BCNT enrolled in the American College of Surgeons National Surgical Quality Improvement Program registry from 2011 to 2015. Multivariate logistic regression was used to determine the impact of select medical and operative factors on the primary outcomes of readmission and reoperation within 30 days, adjusted for relevant covariates. RESULTS We identified 996 patients who underwent resection of a BCNT. The most frequent major complications were readmission (11%), reoperation (8%), surgical site infections (2.6%), and venous thromboembolism (1.5%). The most frequent indications for readmission were management of infection (2.5%), cerebrospinal fluid leak (2.3%), and hydrocephalus (0.5%). Repair of cranial and meningeal defects (3.0%), correction of lagophthalmos (1.2%), and repair of middle ear defects (1.0%) were the most common indications for reoperation. Logistic regression revealed that extremes of age were associated with readmission, whereas preoperative steroid usage, long operative time, and postoperative length of stay >3 days were associated with reoperation (P < 0.05). Obesity trended toward an association with readmission and reoperation. CONCLUSIONS Extremes of age were associated with readmission; preoperative steroid use, long operative time, and postoperative length of stay >3 days were associated with reoperation. Surgeons should consider these factors when assessing risk of postoperative complications for BCNTs.

Among Children Born Extremely Preterm a Higher Level of Circulating Neurotrophins Is Associated with Lower Risk of Cognitive Impairment at School Age

Objectives To test the hypothesis that higher blood levels of neurotrophic proteins (proteins that support neuronal survival and function) in the first 2 weeks of life are associated with a lower risk of cognitive impairment at 10 years. Study design We evaluated 812 10‐year‐old children with neonatal blood specimens enrolled in the multicenter prospective Extremely Low Gestational Age Newborn Study, assessing 22 blood proteins collected on 3 days over the first 2 weeks of life. Using latent profile analysis, we derived a cognitive function level based on standardized cognitive and executive function tests. We defined high exposure as the top quartile neurotrophic protein blood level on ≥2 days either for ≥4 proteins or for a specific cluster of neurotrophic proteins (defined by latent class analysis). Multinomial logistic regression analyzed associations between high exposures and cognitive impairment. Results Controlling for the effects of inflammatory proteins, persistently elevated blood levels of ≥4 neurotrophic proteins were associated with reduced risk of moderate (OR, 0.35; 95% CI, 0.18‐0.67) and severe cognitive impairment (OR, 0.22; 95% CI, 0.09‐0.53). Children with a cluster of elevated proteins including angiopoietin 1, brain‐derived neurotrophic factor, and regulated upon activation, normal T‐cell expressed, and secreted had a reduced risk of adverse cognitive outcomes (OR range, 0.31‐0.6). The risk for moderate to severe cognitive impairment was least with 0‐1 inflammatory and >4 neurotrophic proteins. Conclusions Persisting elevations of circulating neurotrophic proteins during the first 2 weeks of life are associated with lowered risk of impaired cognition at 10 years of age, controlling for increases in inflammatory proteins.