Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation.

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.

The Diagnostic Accuracy of Carotid Doppler in Detecting Anechoic Thrombus Against CT Angiography as the Gold Standard.

Objective In this study, we aimed to assess the diagnostic accuracy of carotid Doppler ultrasound (CDU) in detecting anechoic carotid artery thrombus when compared to CT angiography (CTA) as the gold standard. Materials and methods This prospective comparative study was conducted at the Radiology Department of the Pakistan Institute of Medical Sciences, Islamabad from January 2022 to May 2022. The study enrolled 32 patients who met the inclusion criteria. We evaluated patients admitted to the neurology ward/OPD who were referred to radiology as part of a stroke workup based on their clinical examination and medical history. In all patients, CDU was used to detect free-floating thrombus (FFT)/anechoic thrombus. CTA was used as the gold standard to assess the diagnostic accuracy of CDU. Results The mean age of the study participants was 45.63 ± 7.05 years (range: 33-59 years). Out of 32 patients, 19 (59.4%) were male and 13 (40.6%) were female. The results of CDU were confirmed by CTA in all patients. The diagnostic accuracy of CDU was 53.12% for detecting FFT. The values for sensitivity (54.55%), specificity (50%), positive predictive value (PPV, 70.59%), and negative predictive value (NPV, 33.33%) were also calculated. Conclusion Despite the limited sample size, the study concludes that CDU has a diagnostic accuracy of 53%. CTA still remains the gold standard imaging modality for anechoic thrombus if strong clinical suspicion is present.

Retrospective audit of a convenience cohort of individuals on HIV pre-exposure prophylaxis in Alberta, Canada.

BACKGROUND: We undertook an audit of a province-wide HIV pre-exposure prophylaxis (PrEP) program in Alberta (Canada). METHODS: A retrospective record review of individuals accessing PrEP in Alberta included demographics, PrEP indication(s), and reported non-prescription drug and alcohol use from March 2016 to June 2019. Hepatitis A, B, C, HIV and syphilis serology, serum creatinine, and nucleic acid amplification tests testing for chlamydia and gonorrhea were collected. Descriptive statistics, incidence, and prevalence were calculated. RESULTS: A total of 511 participants were seen at STI, sexual, and reproductive health clinics and private family practitioner (FP) offices; 98.4% (503) were men, median age was 34 years (IQR 28-43 years), and 89.8% (459) were gay or bisexual men who have sex with men. Non-prescription drug use was reported by 39.3% (201) and alcohol use by 55.4% (283). 94.3% (482) reported condomless anal sex in the past 6 months. Testing rates were high (>95%) for all tests except for chlamydia and gonorrhea at the first follow-up visit 89.6%; (3-4 months). There was one HIV seroconversion. The incidence of new bacterial STIs was high: chlamydia 17 cases per 100 person-years (95% CI 13.5% to 21.4%), gonorrhea 11.14 cases per 100 person-years (95% CI 8.3% to 15.0%), and syphilis 1.94 cases per 100 person-years (95% CI 0.73% to 5.12%). CONCLUSIONS: Following implementation of a provincial program for PrEP in Alberta, PrEP initiation and continuation was feasible in a range of settings and by both specialists and FPs.

HISTORIQUE: Les chercheurs ont entrepris une vérification du programme provincial de prophylaxie pré-exposition (PrEP) du VIH en Alberta, au Canada. MÉTHODOLOGIE: Les chercheurs ont procédé à une analyse rétrospective des dossiers des personnes qui ont eu accès à la PrEP en Alberta, y compris les données démographiques, les indications d'administrer une PrEP et la consommation déclarée de médicaments sans ordonnance et d'alcool entre mars 2016 et juin 2019. Ils ont recueilli la sérologie de l'hépatite A, B et C, du VIH et de la syphilis, la créatinine sérique et les tests d'amplification des acides nucléiques de la Chlamydia et de la gonorrhée. Ils ont également calculé les statistiques descriptives, l'incidence et la prévalence de ces maladies. RÉSULTATS: Au total, 511 participants ont été vus dans des cliniques d'ITS, de santé sexuelle et de santé reproductive ainsi qu'au cabinet de médecins de famille privés, soit 98,4 % d'hommes (503), d'un âge médian de 34 ans (ÉIQ : 28 à 43 ans) et 89,8 % (459) d'hommes gay ou bisexuels qui avaient des relations sexuelles avec d'autres hommes. Ainsi, 39,3 % (201) ont déclaré consommer des médicaments sans ordonnance et 55,4 % (283), de l'alcool. De plus, 94,3 % (482) ont indiqué avoir eu des relations sexuelles anales sans préservatif au cours des six mois précédents. Les taux de dépistage étaient élevés (>95 %) à l'égard de tous les tests au premier rendez-vous de suivi (au bout de trois à quatre mois), sauf ceux de la Chlamydia et de la gonorrhée, qui s'élevaient à 89,6 %. Un cas de séroconversion du VIH a été constaté. L'incidence de nouvelles ITS bactérienne était élevée : 17 cas de Chlamydia par 100 années-personnes (IC à 95 %, 13,5 % à 21,4 %), 11,14 cas de gonorrhée par 100 années-personnes (IC à 95 %, 8,3 % à 15,0 %) et 1,94 cas de syphilis par 100 années-personnes (IC à 95 %, 0,73 % à 5,12 %). CONCLUSIONS: Après la mise en œuvre d'un programme provincial de PrEP en Alberta, il a été établi qu'il était possible d'entreprendre et de poursuivre la PrEP dans divers milieux, à l'instigation de spécialistes tout autant que de médecins de famille.

In vitro examination of anti-parasitic, anti-Alzheimer, insecticidal and cytotoxic potential of Ajuga bracteosa Wallich leaves extracts.

This research study is mainly focused to evaluate the anti-parasitic, insecticidal, cytotoxic and anti-alzheimer potential of various leaf extracts of Ajuga bracteosa Wallich ex Bentham. 04 different extracts were prepared using solvent of different polarity to determine the best candidate for potent bioactivity i.e. n-hexane (NH), Ethyl acetate (EA), Ethanol (EL) and Chloroform (CH). Concentrations of each extracts were made specified for all activities. All extracts were exploited for broad range of biomedical applications including leishmaniasis, in vitro anti-Alzheimer, insecticidal and cytotoxic studies. Our results showed that A. bracteosa n-hexane extract was highly active against Leishmania Tropica with significant inhibition of 58 ± 1.61 for promastigote and 63 ± 2.29 for amastigote at 1000 µg/mL. Furthermore, promising anti-alzheimer activity acetylcholinesterase (AChE) 46 ± 0.83 and butrylcholineterase (BChE) 49 ± 1.17 was noted for n-hexane. The insecticidal potential of these extracts were test against five different insects (Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum, Sitophilus oryze, and Callosobruchus analis). The higest mortality rate of insecticidal activity was recorded by n-hexane followed by Ethyl acetate whereas ethanol extract was found to be less effective against all the test species. Significant cytotoxic potential of each plant sample against Artemia salina thus aware us for further detailed research to find out novel drugs. Based on our results we believe that Ajuga bracteosa could be used to develop as a potential botanical insecticide against different insect and pests, such as aphids as well as an excellent source for the compound isolation as anti-tumor agent.

Diagnostic Accuracy of Different Computed Tomography Signs for Differentiating Between Malignant and Cirrhotic Ascites Keeping Ascitic Fluid Cytology as Gold Standard.

Objective The goal of this research was to define the diagnostic precision of CT signs to distinguish malignant ascites from cirrhotic ascites. Ascitic fluid cytology was kept as the gold standard. Study design This research was a prospective cross-sectional study. Place and duration of the study Participants' recruitment started on July 15, 2021, and the whole study lasted about three months till October 15, 2021, at the Radiology Department of Pakistan Institute of Medical Sciences, Islamabad. Patients and methods A total of 80 patients were included in the research and divided into two groups grounded on the cirrhotic or malignant etiology of the ascites based on their fluid cytology. Ascites volume, relative spread between the lesser sac and greater peritoneal cavity, the wall thickness of gallbladder, density of ascites, parietal peritoneum thickness and degree of its enhancement, and presence of septa and loculations were some of the major CT signs studied. Results The average age of patients included in this study was 36.2 ± 6.67 years (range 29-49 years). Of the 80 patients, 50 (62.5 %) were men, and 30 (37.5 %) were women. CT signs associated with the malignant ascites reported in this study were fluid present in the lesser sac (p = 0.03), peritoneal thickening and degree of its enhancement (p = 0.05), increased ascites density (p= 0.001), and presence of septa and loculations (63.6 % of malignant ascites). However, gallbladder wall thickness did not show any variation between both groups. Conclusion We conclude that in the diagnosis of malignant ascites, CT scan imaging can play a vital role. This research approves and testifies the benefits of indirect signs such as the spread of ascites, increased density of ascites, thickening and enhancement of parietal peritoneum, and ascitic fluid complexity in pointing out malignancy as a cause of ascites.

Single-cell RNA-seq reveals profound monocyte changes in Paediatric Inflammatory Multisystem Syndrome Temporally associated with SARS-CoV-2 infection (PIMS-TS)

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening disease occurring several weeks after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MIS-C has overlapping clinical features with Kawasaki Disease (KD), a rare childhood vasculitis. MIS-C therapy is largely based on KD treatment protocols but whether these diseases share underpinning immunological perturbations is unknown. We performed deep immune profiling on blood samples from healthy children and patients with MIS-C or KD. Acute MIS-C patients had highly activated neutrophils, classical monocytes and memory CD8+ T-cells; increased frequencies of B-cell plasmablasts and CD27-IgD-double-negative B-cells; and increased levels of pro-inflammatory (IL6, IL18, IP10, MCP1) but also anti-inflammatory (IL-10, IL1-RA, sTNFR1, sTNFR2) cytokines. Increased neutrophil count correlated with inflammation,cardiac dysfunction and disease severity. Two days after intravenous immunoglobulin (IVIG) treatment, MIS-C patients had increased CD163 expression on monocytes, expansion of a novel population of immature neutrophils, and decreased levels of pro- and anti-inflammatory cytokines in the blood accompanied by a transient increase in arginase in some patients. Our data show MIS-C and KD share substantial immunopathology and identify potential new mechanisms of action for IVIG, a widely used anti-inflammatory drug used to treat MIS-C, KD and other inflammatory diseases.

Antihyperglycemic effect of methanol extract of Tamarix aphylla L. Karst (Saltcedar) in streptozocin–nicotinamide induced diabetic rats

Objective To evaluate the antihyperglycemic potential of Tamarix aphylla (T. aphylla) leaves in STZ–NIC induced diabetes in Wister Albino rats. Methods Acute toxicity study was done to check the toxicity of T. aphylla (L. Karst) methanol extract. T. aphylla leaves extract was administered intraperitoneally (100 mg, 250 mg and 400 mg/kg body weight per day) to diabetic Wister rats for 21 days. The various parameters were studied including fasting blood glucose levels, haemoglobin and glycosylated haemoglobin. Results The treatment groups with the extract at three dose levels expressively abridged the intensities of blood glucose and Glycosylated Haemoglobin. The earlier detected reduced level of plasma haemoglobin of the diabetic rats was raised to near normalcy with treatment of extract. Conclusions The results of the current study confirm that the leaves extract of T. aphylla are nontoxic and have antidiabetic nature.

A novel AP4M1 mutation in autosomal recessive cerebral palsy syndrome and clinical expansion of AP-4 deficiency.

BACKGROUND: Cerebral palsy (CP) is a heterogeneous neurodevelopmental disorder associated with intellectual disability in one-third of cases. Recent findings support Mendelian inheritance in subgroups of patients with the disease. The purpose of this study was to identify a novel genetic cause of paraplegic CP with intellectual disability in a consanguineous Pakistani family. METHODS: We performed whole-exome sequencing (WES) in two brothers with CP and intellectual disability. Analysis of AP4M1 mRNA was performed using quantitative real-time PCR on total RNA from cultured fibroblasts. The brothers were investigated clinically and by MRI. RESULTS: We identified a novel homozygous AP4M1 mutation c.194_195delAT, p.Y65Ffs*50 in the affected brothers. Quantitative RT-PCR analysis showed markedly reduced AP4M1 mRNA levels suggesting partial non-sense mediated mRNA decay. Several clinical and MRI features were consistent with AP-4 complex deficiency. However, in contrast to previously reported cases with AP4M1 mutations our patients show an aggressive behavior and a relatively late onset of disease. CONCLUSION: This study shows an AP4M1 mutation associated with aggressive behavior in addition to mild dysmorphic features, intellectual disability, spastic paraparesis and reduced head circumference. Our findings expand the clinical spectrum associated with AP-4 complex deficiency and the study illustrates the importance of MRI and WES in the diagnosis of patients with CP and intellectual disability.

Typhoid fever in paediatric patients in Quetta, Balochistan, Pakistan.

OBJECTIVES: To determine the seropositivity of typhoid fever in febrile pediatric patients presenting to tertiary care center. METHODS: This observational study was conducted at Children Hospital Quetta (CHQ) from July 2011 to March 2012. The children with three or more days fever, no obvious focus of infection and clinically suspected of typhoid fever were screened. Sterile Blood samples were obtained from febrile patients and Widal and Typhidot® tests were performed for the diagnosis of Typhoid fever in the suspected populations. RESULTS: Total of 2964 clinically suspected patients were screened for typhoid fever. Of these, 550 (18.6%) patients were positive serologically. The higher prevalence of the disease in hot summer season and increasing pattern of the disease was observed in summer days. The disease was higher in school age children under 5-10 years. Although non-significant association was observed on sex basis. CONCLUSION: The findings highlight the considerable burden of typhoid fever in pre-school and school-aged children. The variation in the disease pattern has also been observed under seasonal variation and different age groups, all of which need to be considered in deliberations to control the typhoid fever.

Risk Factors and Phenytoin Prophylaxis for Early Post-Traumatic Seizures among Patients with Traumatic Brain Injury

Background: Post-traumatic seizure is a well-known and serious complication of traumatic brain injury (TBI). The incidence and risk factors vary among study populations. Very little data have been published concerning this in the Malaysian population. The aim of this study was to ascertain the risk factors for the development of early post-traumatic seizures among patients with TBI. Methods: This was a prospective observational study, carried out in Hospital Universiti Sains Malaysia, Kubang Kerian, Kelantan, under the Department of Neurosciences. A total of 157 patients, from all age groups, who were diagnosed with TBI were enrolled from June 2007 to December 2007. They were followed-up for 12 months until death or their first post-traumatic seizure. Survival analysis with Kaplan–Meier curves and Cox proportional hazards regression was performed. Results: A total of 11 (7.0%) of the patients developed early post-traumatic seizures. The risk factors for early post-traumatic seizures were young age (P = 0.021, 95% CI 0.806 to 0.982) and intubated patients (P = 0.029, 95% CI 1.194 to 25.913). The incidence of early post-traumatic seizures in the local population was 7.0%. Conclusion: The incidence of early post-traumatic seizures in the local population of Kelantan and Terengganu is comparable to the incidences reported elsewhere. Younger as well as intubated patients were at a higher risk of developing this condition. It may be necessary to give antiepileptic prophylaxis because any seizure could adversely affect morbidity and mortality. However, the study showed that antiepileptic drug was not beneficial in preventing late post-traumatic seizures, but may have a role in preventing early seizures.