The impact of interval cholecystectomy timing after percutaneous transhepatic cholecystostomy on post-operative adverse outcomes.

OBJECTIVE: This study aims to explore how timing of interval of cholecystectomy (IC) after percutaneous transhepatic cholecystostomy tube (PTC) placement impacts post-operative outcomes. METHODS: A retrospective database analysis of New York State SPARCs database of IC between 2005 and 2015. The timing for IC ranged between > 1 week and < 2 years. Patients undergoing this procedure were further divided into quartiles using 4-time intervals; 1-5 weeks (Q1), 5-8 weeks (Q2), 8-12 weeks(Q3), and > 12 weeks(Q4). The study's primary outcome was hospital length of stay (LOS). Secondary outcomes included discharge status, 30-day readmission, 30-day ED visit, and 90-day reoperation, surgery type, complication, and bile duct injury. Multivariable regression models were used to compare patients across the four-time intervals after adjusting for confounding factors. RESULTS: A total of 1038 patients with a history of PTC followed by IC between > 1 week and < 2 years were included in the final analysis. The median time to IC was 7.7 weeks. Q2 and Q3 both had a significantly higher median LOS of 3 days versus Q1 and Q4 at median of 5 days (p < 0.0001). Patients from racial and ethnic minorities (e.g., African Americans and Hispanics) were more likely to get their IC after 12 weeks (p < 0.05). Further, Black patients had a significantly higher median LOS than White, non-Hispanic patients (8 days vs 4 days, p < 0.0001) and were more likely to have open procedure. Multivariable regression analysis identified shorter LOS during Q2 (Ratio, 0.76, 95%, 0.67-0.87, p < 0.0001), and Q3 (Ratio 0.75, 95% CI, 065-0.86, p < 0.0001) compared to those who got their IC in Q4. Similar findings exist when comparing Q2 and Q3 to those receiving treatment during Q1. CONCLUSION: A time interval of 5-12 weeks between PTC and IC was associated with a decreased LOS. This study also suggests the persistence of racial disparities among these patients.

Percutaneous vertebral augmentation for acute traumatic vertebral Fractures: A TQIP database study.

Patients with vertebral fractures may be treated with percutaneous vertebroplasty (VP) and kyphoplasty (KP) for pain relief. Few studies examine the use of VP and KP in the setting of an acute trauma. In this study, we describe the current use of VP/KP in patients with acute traumatic vertebral fractures. All patients in the ACS Trauma Quality Improvement Program (TQIP) 2016 National Trauma Databank with severe spine injury (spine AIS ≥ 3) met inclusion criteria, including patients who underwent PVA. Logistic regression was used to assess patient and hospital factors associated with PVA; odds ratios and 95 % confidence intervals are reported. 20,769 patients met inclusion criteria and 406 patients received PVA. Patients aged 50 or older were up to 6.73 (2.45 - 27.88) times more likely to receive PVA compared to younger age groups and women compared to men (1.55 [1.23-1.95]). Hospitals with a Level II trauma center and with 401-600 beds were more likely to perform PVA (2.07 [1.51-2.83]) and (1.82 [1.04-3.34]) respectively. African American patients (0.41 [0.19-0.77]), isolated trauma (0.64 [0.42-0.96]), neurosurgeon group size > 6 (0.47 [0.30-0.74]), orthopedic group size > 10, and hospitals in the Northeastern and Western regions of the U.S. (0.33 [0.21-0.51] and 0.46 [0.32-0.64]) were less likely to be associated with PVA. Vertebroplasty and kyphoplasty use for acute traumatic vertebral fractures significantly varied across major trauma centers in the United States by multiple patient, hospital, and surgeon demographics. Regional and institutional practice patterns play an important role in the use of these procedures.

Home Medication Regimens Increase in Complexity After Admission for Fall in the Older Trauma Patient.

BACKGROUND: Hospitalization for the older trauma patient is an opportunity to assess polypharmacy. We hypothesized that medication regimen complexity (RxCS) and pain medication prescriptions (PRxs) would increase in older home-going patients admitted for a fall. METHODS: We retrospectively chart reviewed patients ≥45 years old admitted for a fall at a level 1 trauma center who were discharged home with full medication documentation. RxCS was compared pre-admission and post-discharge with Wilcoxon signed-rank tests; opioid and non-opioid PRxs were compared with Fisher's exact test, α = .05. RESULTS: 103 patients met inclusion criteria; 58% were ≥65 years old. RxCS (9 [.5-13] to 11 [4.5-15], P < .01) increased on discharge. Opioid PRx rates increased significantly in all age groups. Non-opioid PRx rates increased significantly for patients <65 but not for patients ≥65. CONCLUSIONS: Admission for a fall was associated with increases in RxCS, while PRx changes were age-dependent. Providers should recognize that admissions for older patients who fall after trauma are underutilized opportunities to address polypharmacy in high-risk patients.

Post-Trauma Discharge Instructions: Are We Dropping the Ball?

INTRODUCTION: Complex follow-up plans for polytrauma patients are compiled at the end of hospitalization into discharge instructions. We sought to identify how often patient discharge instructions incorrectly communicated specialist recommendations. We hypothesized that patients with more complex hospitalizations would have more discharge instruction errors (DI-errors). METHODS: We reviewed adult trauma inpatients (March 2017-March 2018), excluding those who left against medical advice or were expected to follow up outside our system. Complex hospitalizations were represented using injury severity (ISS), hospital length of stay (LOS), intensive care unit length of stay (iLOS), and number of consultants (NC). We recorded the type of consultant (surgical or nonsurgical), and consultant recommendations for follow-up. DI-errors were defined as either follow-up necessary but omitted or follow-up not necessary yet present on the instructions. Patients with DI-errors were compared to patients without DI-errors. Groups were compared using Wilcoxon rank sum or chi-square (alpha <.05). RESULTS: We included 392 patients (median age 45 [IQR 26-58], ISS 14 [10-21], LOS 6 [3-11]). 55 patients (14%) had DI-errors. Factors associated with DI-errors included the total number of consultants and use of nonsurgical consultants. ISS, LOS, iLOS, were not associated with DI-errors. CONCLUSION: Common measures of admission complexity were not associated with DI-errors, although the number and type of consultants were associated with DI-errors. Non-surgical specialty consultant recommendations were more likely to be omitted. It is crucial for patients to receive accurate discharge instructions, and systematic processes are needed to improve communication with the patients at discharge.

Symptomatic Colorectal Cancer Is Associated With Stage IV Diagnosis in Two Disparate Populations.

In the United States, individuals of Black/African Ancestry (AA) have a higher incidence and mortality from colorectal cancer (CRC) compared to individuals of White/European Ancestry (EA). In order to develop an approach towards disentangling the complex effects of associated race and socioeconomic factors on CRC outcome, we have conducted a manual chart review of sporadic CRC pathological diagnoses (total n = 334) at an urban public hospital (UH) and a suburban university hospital (SH). There were significant differences between the SH and UH CRC patients with respect to Black/AA race (4.2% vs. 89.1%, p < 0.0001) and Medicaid/Self-pay insurance status (14.9% vs. 85.0%, p < 0.0001). While a higher proportion of newly diagnosed CRC patients presented with metastatic stage 4 CRC at the UH (21%) than the SH (12.5%), only the presence of symptoms was significantly associated with stage 4 CRC (odds ratio, OR 7.94, 95% confidence interval, CI 1.83- 34.54, p = 0.0057) in a multivariable generalized linear model (GLM). The proportion of asymptomatic CRC patients was ~20% at both institutions, suggesting that the UH has contributed to reducing CRC disparities. Initiation of CRC screening at the recommended age at both institutions could reduce the proportion of CRC patients presenting with metastatic spread.

Predictors for Withdrawal of Life-Sustaining Therapies in Patients With Traumatic Brain Injury: A Retrospective Trauma Quality Improvement Program Database Study.

Many patients with severe traumatic brain injuries (TBIs) undergo withdrawal of life-sustaining therapies (WLSTs) or transition to comfort measures, but noninjury factors that influence this decision have not been well characterized. We hypothesized that WLST would be associated with institutional and geographic noninjury factors. All patients with a head Abbreviated Injury Scale score ≥3 were identified from 2016 Trauma Quality Improvement Program data. We analyzed factors that might be associated with WLST, including procedure type, age, sex, race, insurance, Glasgow Coma Scale score, mechanism of injury, geographic region, and institutional size and teaching status. Adjusted logistic regression was performed to examine factors associated with WLST. Sixty-nine thousand fifty-three patients were identified: 66% male, 77% with isolated TBI, and 7.8% had WLST. The median age was 56 years (34-73). A positive correlation was found between increasing age and WLST. Women were less likely to undergo WLST than men (odds ratio 0.91 [0.84-0.98]) and took more time to for WLST (3 vs 2 days, P < .001). African Americans underwent WLST at a significantly lower rate (odds ratio 0.66 [0.58-0.75]). Variations were also discovered based on US region, hospital characteristics, and neurosurgical procedures. WLST in severe TBI is independently associated with noninjury factors such as sex, age, race, hospital characteristics, and geographic region. The effect of noninjury factors on these decisions is poorly understood; further study of WLST patterns can aid health care providers in decision making for patients with severe TBI.

Variations in Discharge Destination Following Severe Traumatic Brain Injury across the United States.

BACKGROUND: Discharge destination after traumatic brain injury (TBI) may be influenced by non-patient factors such as regional or institutional practice patterns. We hypothesized that non-patient factors would be associated with discharge destination in severe TBI patients. METHODS: All patients in the ACS Trauma Quality Improvement Program 2016 data set with severe TBI, defined as head Abbreviated Injury Scale ≥3, were categorized by discharge destination. Logistic regression was used to assess factors associated with each destination; odds ratios and 95% confidence level are reported. Regressions were adjusted for age, gender, race, insurance, GCS, ISS, polytrauma, mechanism, neurosurgical procedure, geographic region, teaching status, trauma center level, hospital size, and neurosurgeon group size. RESULTS: 75,690 patients met inclusion criteria. 51% were discharged to home, 16% to rehab, 14% to SNF, and 11% deceased. Mortality was similar across geographic region, teaching status, and hospital size. Southern patients were more likely to be discharged to home while Northeastern patients were more likely to be discharged to rehab. Treatment by groups of 3 or more neurosurgeons was associated with SNF discharge as was treatment at community or non-teaching hospitals. Patients treated at larger hospitals were less likely to be discharged to rehab and more likely to go to SNF. CONCLUSIONS: Geographic region, neurosurgeon group size, teaching status, and hospital size are significantly associated with variation in discharge destination following severe TBI. Regional and institutional variation in practice patterns may play important roles in recovery for some patients with severe TBI.

Fracture of a 40-mm BIOLOX Delta Femoral Head.

Ceramic bearing surfaces have gained popularity in total hip arthroplasty as a result of the favorable mechanical properties and low wear rates. Despite the recognition as an attractive articulation, problems such as ceramic head fracture persist. Smaller heads and higher body mass indices are touted as risk factors for ceramic head fracture and are often associated with antecedent trauma. We present a case report of an 83-year-old male with a body mass index of 26.7 kg/m2 who suffered a fracture of a 40-mm ceramic femoral head. This occurred atraumatically 5 years from his index surgery. This patient underwent revision total hip arthroplasty which included debridement of ceramic debris and alteration of the bearing surface with femoral head and polyethylene liner exchange.

Variation in neurosurgical intervention for severe traumatic brain injury: The challenge of measuring quality in trauma center verification.

BACKGROUND: Intracranial pressure monitor (ICPm) procedure rates are a quality metric for American College of Surgeons trauma center verification. However, ICPm procedure rates may not accurately reflect the quality of care in TBI. We hypothesized that ICPm and craniotomy/craniectomy procedure rates for severe TBI vary across the United States by geography and institution. METHODS: We identified all patients with a severe traumatic brain injury (head Abbreviated Injury Scale, ≥3) from the 2016 Trauma Quality Improvement Program data set. Patients who received surgical decompression or ICPm were identified via International Classification of Diseases codes. Hospital factors included neurosurgeon group size, geographic region, teaching status, and trauma center level. Two multiple logistic regression models were performed identifying factors associated with (1) craniotomy with or without ICPm or (2) ICPm alone. Data are presented as medians (interquartile range) and odds ratios (ORs) (95% confidence interval). RESULTS: We identified 75,690 patients (66.4% male; age, 59 [36-77] years) with a median Injury Severity Score of 17 (11-25). Overall, 6.1% had surgical decompression, and 4.8% had ICPm placement. Logistic regression analysis showed that region of the country was significantly associated with procedure type: hospitals in the West were more likely to use ICPm (OR, 1.34 [1.20-1.50]), while Northeastern (OR, 0.80 [0.72-0.89]), Southern (OR, 0.84 [0.78-0.92]), and Western (OR, 0.88 [0.80-0.96]) hospitals were less likely to perform surgical decompression. Hospitals with small neurosurgeon groups (<3) were more likely to perform surgical intervention. Community hospitals are associated with higher odds of surgical decompression but lower odds of ICPm placement. CONCLUSION: Both geographic differences and hospital characteristics are independent predictors for surgical intervention in severe traumatic brain injury. This suggests that nonpatient factors drive procedural decisions, indicating that ICPm rate is not an ideal quality metric for American College of Surgeons trauma center verification. LEVEL OF EVIDENCE: Epidemiological, level III; Care management/Therapeutic level III.

Draft Genome Sequences of Staphylococcus Podophages JBug18, Pike, Pontiff, and Pabna.

We report here the draft genome sequences of Staphylococcus bacteriophages JBug18, Pike, Pontiff, and Pabna, which infect and lyse S. epidermidis and S. aureus strains. All bacteriophages belong to the morphological family Podoviridae and constitute attractive candidates for use as whole-phage therapeutics due to their compact genomes and lytic lifestyles.

Dysregulation of the Mitochondrial Unfolded Protein Response Induces Non-Apoptotic Dopaminergic Neurodegeneration in C. elegans Models of Parkinson's Disease.

Due to environmental insult or innate genetic deficiency, protein folding environments of the mitochondrial matrix are prone to dysregulation, prompting the activation of a specific organellar stress-response mechanism, the mitochondrial unfolded protein response (UPRMT). In Caenorhabditis elegans, mitochondrial damage leads to nuclear translocation of the ATFS-1 transcription factor to activate the UPRMT After short-term acute stress has been mitigated, the UPRMT is eventually suppressed to restore homeostasis to C. elegans hermaphrodites. In contrast, and reflective of the more chronic nature of progressive neurodegenerative disorders such as Parkinson's disease (PD), here, we report the consequences of prolonged, cell-autonomous activation of the UPRMT in C. elegans dopaminergic neurons. We reveal that neuronal function and integrity decline rapidly with age, culminating in activity-dependent, non-apoptotic cell death. In a PD-like context wherein transgenic nematodes express the Lewy body constituent protein α-synuclein (αS), we not only find that this protein and its PD-associated disease variants have the capacity to induce the UPRMT, but also that coexpression of αS and ATFS-1-associated dysregulation of the UPRMT synergistically potentiate dopaminergic neurotoxicity. This genetic interaction is in parallel to mitophagic pathways dependent on the C. elegans PINK1 homolog, which is necessary for cellular resistance to chronic malfunction of the UPRMT Given the increasingly recognized role of mitochondrial quality control in neurodegenerative diseases, these studies illustrate, for the first time, an insidious aspect of mitochondrial signaling in which the UPRMT pathway, under disease-associated, context-specific dysregulation, exacerbates disruption of dopaminergic neurons in vivo, resulting in the neurodegeneration characteristic of PD.SIGNIFICANCE STATEMENT Disruptions or alterations in the activation of pathways that regulate mitochondrial quality control have been linked to neurodegenerative diseases due in part to the central role of mitochondria in metabolism, ROS regulation, and proteostasis. The extent to which these pathways, including the mitochondrial unfolded protein response (UPRMT) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to compounding stressors. Furthermore, therapeutic strategies that aim to induce these pathways may benefit from increased study into cellular responses that arise from long-term or ectopic stimulation, especially in neuronal compartments. By demonstrating the detrimental consequences of prolonged cellular activation of the UPRMT, we provide evidence that this pathway is not a universally beneficial mechanism because dysregulation has neurotoxic consequences.

Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties.

BACKGROUND: The availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers. RESULTS: To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (> or = two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach. CONCLUSIONS: In this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.

Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and beta-catenin.

Macrophage colony-stimulating factor (M-CSF) influences the proliferation and survival of mononuclear phagocytes through the receptor CSF-1R. The adaptor protein DAP12 is critical for the function of mononuclear phagocytes. DAP12-mutant mice and humans have defects in osteoclasts and microglia, as well as brain and bone abnormalities. Here we show DAP12 deficiency impaired the M-CSF-induced proliferation and survival of macrophages in vitro. DAP12-deficient mice had fewer microglia in defined central nervous system areas, and DAP12-deficient progenitors regenerated myeloid cells inefficiently after bone marrow transplantation. Signaling by M-CSF through CSF-1R induced the stabilization and nuclear translocation of beta-catenin, which activated genes involved in the cell cycle. DAP12 was essential for phosphorylation and nuclear accumulation of beta-catenin. Our results provide a mechanistic explanation for the many defects of DAP12-deficient mononuclear phagocytes.

Proteomic comparison of Entamoeba histolytica and Entamoeba dispar and the role of E. histolytica alcohol dehydrogenase 3 in virulence.

The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1:IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1:IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1:IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.

Differences in complement-mediated killing of Entamoeba histolytica between men and women--an explanation for the increased susceptibility of men to invasive amebiasis?

Men are more than 7 times more likely to develop amebic liver abscess or amebic dysentery caused by Entamoeba histolytica than women. Because the complement system could play a key role in controlling amebiasis, we determined whether serum from men and women differ in the ability to kill amebic trophozoites. We found that serum from women was significantly more effective in killing E. histolytica trophozoites than serum from men, and this killing was complement dependent. Our results provide a possible explanation for the differential susceptibility of men and women to amebic liver abscess and amebic colitis.

Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression.

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human beta-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

The immunogenetics of smallpox vaccination.

We hypothesized that individuals who develop fever after smallpox vaccination have genetically determined differences in their immune responses to vaccinia virus. We looked for an association between the development of fever and single-nucleotide polymorphisms (SNPs) in 19 candidate genes in 346 individuals previously assessed for clinical responses to smallpox vaccination. Fever after smallpox vaccination is associated with specific haplotypes in the interleukin (IL)-1 gene complex and in the IL18 gene. A haplotype in the IL4 gene was highly significant for reduced susceptibility to the development of fever after vaccination among vaccinia-naive individuals. Our results indicate that certain haplotypes in the IL-1 gene complex and in IL18 and IL4 predict an altered likelihood of the development of fever after smallpox vaccination. Our findings also raise the possibility that these same haplotypes may identify individuals at risk for the development of fever after receipt of other live virus vaccines, providing information that could be useful in anticipating and preventing more-serious adverse events.

Antibody responses to vaccinia membrane proteins after smallpox vaccination.

BACKGROUND: Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins after VV vaccination have not been well characterized. METHODS: Pre- and postvaccination (day 26-30) serum specimens from 80 VV vaccine recipients were examined for immunoglobulin G antibodies specific for B5, A33, A27, and L1 by enzyme-linked immunosorbent assay (ELISA). Responses were compared between vaccinia-naive and previously vaccinated (nonnaive) recipients and between nonnaive recipients of undiluted or 1 : 10 diluted vaccine. RESULTS: VV vaccination elicited anti-A33 and anti-A27 antibodies in nearly all vaccinia-naive subjects (100% and 93%, respectively). Preexisting antibodies were commonly detected in nonnaive subjects (for anti-B5, 68%; for anti-A33, 59%; for anti-A27, 38%; and for anti-L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010 for pre- vs. postvaccination; P<.001), whereas anti-L1 antibody responses were less robust (detection rate, 31%; GMT, 75) in nonnaive subjects. Diluted vaccine elicited antibody responses that were similar to those elicited by undiluted vaccine. CONCLUSIONS: Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status but not with respect to 10-fold dilution of vaccine. B5, A33, and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines.

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

Transcriptomic comparison of two Entamoeba histolytica strains with defined virulence phenotypes identifies new virulence factor candidates and key differences in the expression patterns of cysteine proteases, lectin light chains, and calmodulin.

The availability of Rahman, and the virulent HM-1:IMSS strain of E. histolytica, provides a powerful tool for identifying virulence factors of E. histolytica. Here we report an attempt to identify potential virulence factors of E. histolytica by comparing the transcriptome of E. histolytica HM-1:IMSS and E. histolytica Rahman. With phenotypically defined strains, we compared the transcriptome of Rahman and HM-1:IMSS using a custom 70mer oligonucleotide based microarray that has essentially full representation of the E. histolytica HM-1:IMSS genome. We find extensive differences between the two strains, including distinct patterns of gene expression of cysteine proteinases, AIG family members, and lectin light chains.