rVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.

Predicting the Risk to Develop Preeclampsia in the First Trimester Combining Promoter Variant -98A/C of LGALS13 (Placental Protein 13), Black Ethnicity, Previous Preeclampsia, Obesity, and Maternal Age.

BACKGROUND: LGALS13 (placental protein 13 [PP13]) promoter DNA polymorphisms was evaluated in predicting preeclampsia (PE), given PP13's effects on hypotension, angiogenesis, and immune tolerance. METHODS: First-trimester plasma samples (49 term and 18 intermediate) of PE cases matched with 196 controls were collected from King's College Hospital, London, repository. Cell-free DNA was extracted and the LGALS13 exons were sequenced after PCR amplification. Expression of LGALS13 promoter reporter constructs was determined in BeWo trophoblast-like cells with luciferase assays. Adjusted odds ratio (OR) was calculated for the A/A genotype combined with maternal risk factors. RESULTS: The A/A, A/C, and C/C genotypes in the -98 promoter position were in Hardy-Weinberg equilibrium in the control but not in the PE group (p < 0.036). The dominant A/A genotype had higher frequency in the PE group (p < 0.001). The A/C and C/C genotypes protected from PE (p < 0.032). The ORs to develop term and all PE, calculated for the A/A genotype, previous PE, body mass index (BMI) >35, black ethnicity, and maternal age >40 were 15.6 and 11.0, respectively (p < 0.001). In luciferase assays, the "-98A" promoter variant had lower expression than the "-98C" variant in non-differentiated (-13%, p = 0.04) and differentiated (-26%, p < 0.001) BeWo cells. Forskolin-induced differentiation led to a larger expression increase in the "-98C" variant than in the "-98A" variant (4.55-fold vs. 3.85-fold, p < 0.001). CONCLUSION: Lower LGALS13 (PP13) expression with the "A" nucleotide in the -98 promoter region position (compared to "C") and high OR calculated for the A/A genotype in the -98A/C promoter region position, history of previous PE, BMI >35, advanced maternal age >40, and black ethnicity could serve to aid in PE prediction in the first trimester.

Evaluation of a new immunochromatography test for rapid and simultaneous detection of Clostridium difficile antigen and toxins.

BACKGROUND: Clostridium difficile infection is considered the most common cause of nosocomial infectious diarrhea among adults in the developed world. It is responsible for virtually all cases of pseudomembranous colitis. The Tox A/B enzyme immunoassay (EIA) is the most widely used test for the detection of C. difficile toxins A and B. However, it is associated with poor sensitivity and an unacceptable high rate of false-negative results. OBJECTIVES: To evaluate the performance of the C. DIFF QUIK CHEK COMPLETE assay, designed to simultaneously detect C. difficile-produced glutamate dehydrogenase (GDH) and toxins A and B. METHODS: Using the C. DIFF QUIK CHEK COMPLETE assay, the Tox A/B EIA, and polymerase chain reaction (PCR), we tested 223 stool specimens from hospitalized patients with antibiotics-associated diarrhea. Sensitivity and specificity, and positive and negative predictive values (PPV, NPV) were calculated for the C. DIFF QUIK CHEK COMPLETE test and the Tox A/B EIA against PCR RESULTS: The C. DIFF QUIK CHEK COMPLETE test had a sensitivity of 83.5% and specificity of 94.3% compared to PCR for Tox A/B, with 93.7% correlation (PPV 98.5%, NPV 91.7%). The Tox A/B EIA yielded corresponding values of 72.1% and 93.1%, with 85.6% correlation (PPV 85.1%, NPV 85.8%). CONCLUSIONS: Given the importance of an early and appropriate diagnosis of Clostridium difficile-associated infection, the C. DIFF QUIK CHEK COMPLETE test may be of huge benefit to practitioners.

Unusual "flesh-eating" strains of Escherichia coli.

Monomicrobial necrotizing fasciitis (type II) is typically caused by group A streptococcus alone or in combination with Staphylococcus aureus. Escherichia coli has been isolated from polymicrobial or Fournier's gangrene but has rarely been reported in monomicrobial necrotizing fasciitis. We describe the clinical characteristics and outcomes of seven cases of monomicrobial E. coli necrotizing fasciitis and/or severe soft tissue infection diagnosed at a single institution during an 18-month period. Four isolates from three patients and two isolates from two patients with type I polymicrobial severe soft tissue infection (controls) were assayed by the randomly amplified polymorphic DNA (RAPD) analysis for fingerprinting and PCR amplification of primers in order to detect cytotoxic necrotizing factor 1 and 2 (cnf1 and cnf2) genes. All patients had some type of immune suppression. The limb was the most commonly involved organ. In all cases, E. coli was isolated as a monomicrobial pathogen from blood, fascia, or both. All patients died during hospitalization, three within the first 48 h. The RAPD amplification assay showed a high degree of genetic diversity among the "flesh-eating" strains and controls. The cnf1 toxin gene was identified in two out of three cases, but not in the controls. cnf2 was not detected in any of the patients. E. coli may be responsible for life-threatening necrotizing fasciitis. Further research is needed to reveal relevant risk factors, reservoirs, and modes of transmission of cnf1 E. coli.

Molecular epidemiology of clostridium difficile in a tertiary medical center in Israel: emergence of the polymerase chain reaction ribotype 027.

BACKGROUND: The rate of infection with Clostridium difficile colitis and its associated mortality have been increasing in the last decade. The molecular epidemiology of C. difficile in Israel has not been studied. OBJECTIVES: To screen for the existence of the 027 and 078 ribotypes and determine the longitudinal molecular epidemiology of the circulating clinical C. difficile isolates in a large hospital in central Israel. METHODS: Polymerase chain reaction (PCR) ribotyping was performed on C. difficile isolates obtained from hospitalized patients from November 2003 to May 2004 (first study period) and September 2009 (second study period). Isolates with PCR ribotype patterns, unlike those of the available reference strains (078 and 027), were labeled with letters. Forty-six isolates from the first study period and 20 from the second were analyzed. RESULTS: PCR strain typing of C. difficile isolates yielded approximately 26 unique ribotypes. During the first study period, ribotype A and B accounted for 30% and 28%, respectively, whereas ribotype E and K accounted for 6.5% for each. During the second study period, ribotypes A, E and K disappeared, and the incidence of ribotype B decreased from 28% to 15%. One isolate (1/20, 5%) emerged during the second period and was identified as ribotype 027. Moxifloxacin resistance was found in 93% of ribotype A isolates, 81% of the ribotype B group, and in 44% of other ribotypes. CONCLUSIONS: The predominant ribotypes circulating in our institution were diverse and changing. This is the first report on the emergence of the 027 ribotype in Israel.

Projection of Gut Microbiome Pre- and Post-Bariatric Surgery To Predict Surgery Outcome.

Bariatric surgery is often the preferred method to resolve obesity and diabetes, with ∼800,000 cases worldwide yearly and high outcome variability. The ability to predict the long-term body mass index (BMI) change following surgery has important implications for individuals and the health care system in general. Given the tight connection between eating habits, sugar consumption, BMI, and the gut microbiome, we tested whether the microbiome before any treatment is associated with different treatment outcomes, as well as other intakes (high-density lipoproteins [HDL], triglycerides, etc.). A projection of the gut microbiome composition of obese (sampled before and after bariatric surgery) and lean patients into principal components was performed, and the relation between this projection and surgery outcome was studied. The projection revealed three different microbiome profiles belonging to lean, obese, and obese individuals who underwent bariatric surgery, with the postsurgery microbiome more different from the lean microbiome than the obese microbiome. The same projection allowed for a prediction of BMI loss following bariatric surgery, using only the presurgery microbiome. The microbial changes following surgery were an increase in the relative abundance of Proteobacteria and Fusobacteria and a decrease in Firmicutes. The gut microbiome can be decomposed into main components depicting the patient's development and predicting in advance the outcome. Those may be translated into the better clinical management of obese individuals planning to undergo metabolic surgery. IMPORTANCE BMI and diabetes can affect the gut microbiome composition. Bariatric surgery has large variabilities in the outcome. The microbiome was previously shown to be a good predictor for multiple diseases. We analyzed here the gut microbiome before and after bariatric surgery and showed the following. (i) The microbiome before surgery can be used to predict surgery outcomes. (ii) The postsurgery microbiome drifts further away from the lean microbiome than the microbiome of the presurgery obese patients. These results can lead to a microbiome-based presurgery decision whether to perform surgery.

Group B Streptococcus serotypes associated with different clinical syndromes: Asymptomatic carriage in pregnant women, intrauterine fetal death, and early onset disease in the newborn.

OBJECTIVES: To study Group B Streptococcus (GBS) isolates associated with different clinical syndromes: asymptomatic carriage in pregnant women, intrauterine fetal death (IUFD), and early onset disease (EOD) in the newborn. METHODS: GBS isolates were collected from asymptomatic pregnant women admitted for labor, IUFD cases, and neonates with EOD. Serotypes and antibiotic susceptibilities were determined. Multilocus sequence typing (MLST) was performed to assess genetic epidemiology. RESULTS: GBS carriage rate was 26.1% (280/1074). The dominant serotype among asymptomatic pregnant women was VI [98/240 women (40.8%)], followed by serotypes III, V and IV in 42/240 (17.5%), 30/240 (12.5%) and 28/240 (11.7%) women, respectively. The dominant serotype in IUFD cases was serotype VI [10/13 (76.9%)]. In contrast the prevalent serotype among EOD cases was III [16/19 (84.2%)]. ST-1 was associated with IUFD [7/13 (53.8%)], ST-17 was associated with serotype III and EOD in the newborn 14/19 (73.7%)]. Erythromycin and clindamycin resistance reached 36.8%, 7.7% and 20.0%among EOD, vaginal carriage and IUFD, respectively. CONCLUSIONS: Serotypes VI and ST-1 were dominant among asymptomatic pregnant women and in IUFD cases while EOD was associated with serotype III and ST-17. Invasive mechanisms thus may differ between IUFD and EOD in the newborn and virulence may be related to capsule serotype. Resistance rates to erythromycin and clindamycin were high in EOD cases.

Evaluation of CHROMagar KPC for rapid detection of carbapenem-resistant Enterobacteriaceae.

A new CHROMagar KPC medium was compared to MacConkey agar with carbapenem discs and PCR for the bla(KPC) gene for rapid detection of carbapenem-resistant Klebsiella pneumoniae. The sensitivity and specificity relative to PCR were 100% and 98.4%, respectively, for CHROMagar KPC and 92.7% and 95.9%, respectively, for MacConkey agar.

Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel.

This report describes an outbreak of carbapenem-resistant KPC-3-producing Klebsiella pneumoniae outside the USA. Ninety patients from different departments of a tertiary medical centre were diagnosed with carbapenem-resistant, extended-spectrum beta-lactamase (ESBL)-negative Klebsiella pneumoniae infection by standard methods over a 10-month period in 2006. Fifteen randomly selected outbreak isolates were subjected to randomly amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) as well as PCR amplification and sequencing of the KPC genes, and the findings were compared with two carbapenem-susceptible K. pneumoniae isolates (one ESBL-positive and one ESBL-negative). All the outbreak isolates were resistant to all fluoroquinolones and beta-lactam antibiotics tested, including carbapenems, and were sensitive only to colistin, gentamicin and most of them also to tigecycline. On RAPD-PCR, all 15 outbreak isolates were identical to each other and clearly distinguishable from control strains, indicating clonality. The KPC-3 enzyme was identified by nucleotide sequencing analysis in all outbreak isolates but not in the control strains. These findings should alert government and medical authorities to institute stringent control measures and to initiate research into therapeutic and preventive strategies.

Developing a new algorithm for first and second trimester preeclampsia screening in twin pregnancies.

OBJECTIVES: Construct a new preeclampsia predicting algorithm in twins. METHODS: Twins sampled at 10-13 and 16-20 gestational weeks and their marker values were log transformed into multiples of the gestation-specific medians (MoMs) for singletons and entered into a new logistic regression model with/without prior risk factors. RESULTS: The cohort included 9 PE (18 samples) and 96 unaffected cases (175 samples) twin pregnant women. The algorithm constructed of PlGF, PAPP-A, PP13, Doppler UTPI, and MAP with prior risk factors generated an area under the curve of 0.918, 75% detection rate for 10% false-positive rate. CONCLUSIONS: The algorithm effectively forecasted twin risk to develop PE.

Celecoxib leads to G2/M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner.

A unique in vitro system has been developed in our lab that consists of normal enterocytes derived from the rat ileum (IEC-18 cells) and their transformed derivatives with c-K-ras (R1 cells), anti-sense bak (B3 cells) and cyclin D1 (D1 cells). R1 and B3 cells express high level of COX-2 protein and PGE2. IEC 18 and D1 cells express negligible amount of COX-2, and produce very low level of PGE2. A relatively low dose of celecoxib (5-10 microM) induced G2/M arrest, followed by induction of apoptosis in the transformed but not in the normal cells. Down-regulation of cyclin B1 and up-regulation of p21 expressions independent of p53 might have cause this cell cycle block. Growth inhibition was related to COX-2 function with 90-95% reduction in PGE2 production. These findings may be of clinical importance, since low concentration of celecoxib can be achieved in human serum following standard anti-inflammatory (100-200 mg bid) regime.

Celecoxib and curcumin synergistically inhibit the growth of colorectal cancer cells.

PURPOSE: Multiple studies have indicated that cyclooxygenase-2 (COX-2) inhibitors may prevent colon cancer, which is one of the leading causes of cancer death in the western world. Recent studies, however, showed that their long-term use may be limited due to cardiovascular toxicity. This study aims to investigate whether curcumin potentiates the growth inhibitory effect of celecoxib, a specific COX-2 inhibitor, in human colon cancer cells. EXPERIMENTAL DESIGN: HT-29 and IEC-18-K-ras (expressing high levels of COX-2), Caco-2 (expressing low level of COX-2), and SW-480 (no expression of COX-2) cell lines were exposed to different concentrations of celecoxib (0-50 micromol/L), curcumin (0-20 micromol/L), and their combination. COX-2 activity was assessed by measuring prostaglandin E(2) production by enzyme-linked immunoassay. COX-2 mRNA levels were assessed by reverse transcription-PCR. RESULTS: Exposure to curcumin (10-15 micromol/L) and physiologic doses of celecoxib (5 micromol/L) resulted in a synergistic inhibitory effect on cell growth. Growth inhibition was associated with inhibition of proliferation and induction of apoptosis. Curcumin augmented celecoxib inhibition of prostaglandin E(2) synthesis. The drugs synergistically down-regulated COX-2 mRNA expression. Western blot analysis showed that the level of COX-1 was not altered by treatment with celecoxib, curcumin, or their combination. CONCLUSIONS: Curcumin potentiates the growth inhibitory effect of celecoxib by shifting the dose-response curve to the left. The synergistic growth inhibitory effect was mediated through a mechanism that probably involves inhibition of the COX-2 pathway and may involve other non-COX-2 pathways. This synergistic effect is clinically important because it can be achieved in the serum of patients receiving standard anti-inflammatory or antineoplastic dosages of celecoxib.

Conservation of expression and alternative splicing in the prosaposin gene.

Prosaposin is the precursor of four lysosomal activator molecules known as saposins A, B, C and D. It is also secreted and was proposed to be a neurotrophic factor. The neurotrophic function was attributed to the amino terminus of saposin C. In man, mouse and rat prosaposin is transcribed to two major isoforms differing in the inclusion of 9 bps of exon 8 within the saposin B domain. In the present study, we show that there is evolutionary conservation of the prosaposin structure and alternative splicing in chick and zebrafish as well. Moreover, there is conservation in prosaposin expression as tested immunohistochemically in the mouse and chick developing brain. We developed a sensitive assay to quantitate the prosaposin alternatively spliced forms. Our results indicate that, in mouse brain, skeletal and cardiac muscle the exon 8-containing RNA is most abundant, while it is almost absent from visceral and smooth muscle-containing organs. We observed temporal and differential expression of the alternatively spliced prosaposin mRNAs in mouse and chick brain as well as during development. The elevation in the abundance of exon 8-containing prosaposin RNA during mouse and chick brain development may suggest a role for the exon 8-containing prosaposin form in this process.

Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis.

The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen.

Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations.

The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF-induced growth arrest occurs at the G1 phase, accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown by DNA array screening and confirmed for some by immunoblotting. These genes include regulators of cell differentiation and proliferation such as c-jun, JunD, cyclin-D1, NFkappaB1 and of plasma-membrane microdomain morphology, such as ezrin. The transcription factor Id1 is downregulated, consistent with the cells' exit from the mitotic cycle. Moreover, following FGF stimulation, levels of FGFR3 mRNA and protein decline, as does downstream signaling through the MAPK pathway. The importance of this FGFR3-mediated on-off control is illustrated in transgenic mice expressing mutant, hyperactive FGFR3, where abnormally high levels of NFkappaB are expressed throughout their bone growth-plates. A working model is presented of the signaling network involved in regulating FGF-induced chondrocyte differentiation and receptor downregulation.