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Current Protocols Jan 2022Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths...
Entamoeba histolytica is a parasitic protozoan and the causative agent of amoebiasis in humans. Amoebiasis has a high incidence of disease, resulting in ∼67,900 deaths per year, and it poses a tremendous burden of morbidity and mortality in children. Despite its importance, E. histolytica is an understudied parasite. These protocols describe the in vitro growth, maintenance, cryopreservation, genetic manipulation, and cloning of axenic E. histolytica trophozoites. There has been significant progress in genetic manipulation of this organism over the past decade, and these protocols outline the ways in which these advances can be implemented. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Culturing E. histolytica trophozoites Support Protocol 1: Preparation of TYI-S-33 medium Support Protocol 2: Lot testing of Biosate peptone and adult bovine serum for TYI-S-33 medium Basic Protocol 2: Cryopreservation of E. histolytica trophozoites Support Protocol 3: Preparation of cryoprotectant solutions Basic Protocol 3: Transfection of E. histolytica trophozoites with Attractene reagent Basic Protocol 4: Creating clonal lines using limiting dilution Basic Protocol 5: Knockdown of one to two genes with trigger-induced RNA interference Support Protocol 4: Evaluation of RNA interference knockdown with reverse transcriptase PCR Basic Protocol 6: E. histolytica growth curves.
Topics: Adult; Animals; Child; Culture Media; Entamoeba histolytica; Genetic Techniques; Humans; RNA Interference; Trophozoites
PubMed: 35085418
DOI: 10.1002/cpz1.327 -
Current Protocols Feb 2024Anesthesia and analgesia play pivotal roles in ethically and humanely using animal models in research, especially concerning mice and rats. These rodent species,...
Anesthesia and analgesia play pivotal roles in ethically and humanely using animal models in research, especially concerning mice and rats. These rodent species, extensively utilized in scientific investigations due to their genetic resemblance to humans, serve as invaluable tools for studying diseases and testing treatments. Proper anesthesia and analgesia not only prioritize animal welfare but also heighten experimental validity by minimizing stress-induced physiological responses. Recent years have seen remarkable advancements in anesthesia for mice and rats. The focus has shifted away from the 'one size fits all' toward tailoring anesthesia protocols, considering factors like age, strain, and the nature of the experimental procedure. The use of inhalation agents such as isoflurane and sevoflurane is often preferred due to their rapid induction and recovery characteristics, allowing precise control over anesthesia depth. However, refinements in injectable anesthetic agents also provide researchers the flexibility to select suitable agents based on study requirements. Additionally, progress in analgesic techniques has led to effective pain management strategies for these rodents. Common analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and local anesthetics are administered to alleviate pain and discomfort. However, standard practice also involves continuous monitoring of animals' behavior and physiological parameters, ensuring timely adjustments in analgesic regimens for optimal pain relief without compromising experimental outcomes. By integrating tailored anesthesia and analgesia protocols into the experimental design, researchers uphold high animal welfare standards while obtaining reliable scientific data. This contributes significantly to advancing medical knowledge and therapeutic interventions with reproducible results. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Injectable anesthesia for mouse and rat Basic Protocol 2: Inhalant anesthesia using isoflurane for mouse and rat Basic Protocol 3: Analgesia for mice and rats.
Topics: Humans; Rats; Mice; Animals; Pain Management; Isoflurane; Anesthesia; Pain; Analgesia; Analgesics; Anesthetics, Local
PubMed: 38406895
DOI: 10.1002/cpz1.995 -
Critical Reviews in Oncology/hematology Aug 2023Treatment of multiple myeloma (MM) has seen great advances in recent years, and a key contributor to this change has been the effective use of combination therapies,... (Review)
Review
Treatment of multiple myeloma (MM) has seen great advances in recent years, and a key contributor to this change has been the effective use of combination therapies, which have improved both the depth and duration of patient responses. Immunomodulatory drug (IMiD) agents (lenalidomide and pomalidomide) have both tumoricidal and immunostimulatory functions, and due to their multiple mechanisms of action have become the backbone of numerous combination treatments in the newly diagnosed and relapsed/refractory settings. Although IMiD agent-based combination regimens provide improved clinical outcomes for patients with MM, the mechanisms underpinning these combinations are not well understood. In this review we describe the potential mechanisms of synergy leading to the enhanced activity observed when IMiD agents and other drug classes are used in combination through interrogation of the current knowledge surrounding their mechanism of actions.
Topics: Humans; Multiple Myeloma; Lenalidomide; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Immunomodulation
PubMed: 37268176
DOI: 10.1016/j.critrevonc.2023.104041 -
Current Protocols Feb 2021Transformation techniques used to genetically manipulate Borrelia burgdorferi, the agent of Lyme disease, play a critical role in generating mutants that facilitate...
Transformation techniques used to genetically manipulate Borrelia burgdorferi, the agent of Lyme disease, play a critical role in generating mutants that facilitate analyses of the role of genes in the pathophysiology of this bacterium. A number of borrelial mutants have been successfully isolated and characterized since the first electrotransformation procedure was established 25 years ago (Samuels, 1995). This article is directed at additional considerations for transforming infectious B. burgdorferi to generate strains retaining the plasmid profile of the parental strain, enabling analysis of transformants for in vitro and in vivo phenotypes. These methods are built on previously published protocols and are intended to add steps and tips to enhance transformation efficiency and recovery of strains amenable for studies involving colonization, survival, and transmission of B. burgdorferi during the vector and vertebrate phases of infection. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of stock cultures, propagation of spirochetes, and analysis of plasmid profiles Basic Protocol 2: Preparation of plasmid and linear DNA templates for transformation Basic Protocol 3: Transformation of B. burgdorferi Basic Protocol 4: Antibiotic selection of borrelial transformants Basic Protocol 5: Isolation of borrelial transformants in agar overlays Basic Protocol 6: Complementation of mutant borrelial strains in cis or in trans.
Topics: Borrelia burgdorferi; Humans; Lyme Disease; Plasmids
PubMed: 33661557
DOI: 10.1002/cpz1.61 -
Journal of Dairy Science Jun 2023Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use... (Review)
Review
Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.
Topics: Female; Cattle; Animals; Milk; Mastitis, Bovine; Anti-Infective Agents; Lactation; Mammary Glands, Animal; Cell Count; Anti-Bacterial Agents; Cattle Diseases
PubMed: 37080782
DOI: 10.3168/jds.2022-22826 -
Open Veterinary Journal Sep 2023Canine lymphoma is one of the most commonly reported hematopoietic tumors.
BACKGROUND
Canine lymphoma is one of the most commonly reported hematopoietic tumors.
AIM
A few retrospective studies have involved complex evaluations including diagnostic features and treatment protocols, but these studies infrequently demonstrate variable factors that affect survival time, and comparisons among chemotherapeutic protocols are limited. This study aimed to identify prognostic factors that can be simply detected in dogs with lymphoma, such as abnormalities in physical and hematologic findings, and treatment protocols.
METHODS
Clinical records of 77 dogs diagnosed with lymphoma were retrospectively reviewed.
RESULTS
The author newly identified leukocyte and platelet abnormalities as negative prognostic factors. Furthermore, this study suggests that decreased gastrointestinal toxicity and improvements of hematologic abnormalities, such as anemia, thrombocytopenia, and lymphocytosis or lymphoblasts, in peripheral blood during chemotherapy act as positive prognostic factors. Finally, strict adherence to therapeutic protocol and selecting multiple agents as rescue protocol are important to prolong survival time.
CONCLUSION
This study identified indicators to be used as prognostic factors through survival analysis.
Topics: Dogs; Animals; Retrospective Studies; Lymphoma; Thrombocytopenia; Antineoplastic Combined Chemotherapy Protocols; Survival Analysis; Dog Diseases
PubMed: 37842100
DOI: 10.5455/OVJ.2023.v13.i9.8 -
International Dental Journal Oct 2022The aim of this work was to review the protocol of the use of silver diamine fluoride (SDF) for arresting caries, specifically the application time. (Review)
Review
OBJECTIVE
The aim of this work was to review the protocol of the use of silver diamine fluoride (SDF) for arresting caries, specifically the application time.
METHOD
Two researchers searched manufacturers' instructions, YouTube videos, and 5 databases (Embase, Medline, PubMed, Scopus, and Web of Science). Manufacturers' instructions, videos from national dental organisations, and peer-reviewed journal articles that published the SDF application protocol in English for arresting caries were selected.
RESULTS
The review included 14 protocols from 15 publications from 4 manufacturers, 3 dental associations, and 7 author teams (one team had 2 articles). The American Dental Association and the British Society of Paediatric Dentistry provided their SDF application protocols on YouTube. The American Academy of Paediatric Dentistry and 7 author teams published their protocols in journal articles. Seven publications suggested an SDF application time of 60 seconds. Seven publications suggested a time range of 10 seconds to 240 seconds. Two publications suggested caries excavation, but 4 publications suggested no caries excavation before SDF application. The procedures from at least 5 publications involved protecting the gingiva with petroleum jelly, isolating the carious tooth with cotton rolls, drying the carious lesion with a 3-in-1 syringe, applying SDF solution with a micro brush for 60 seconds, removing excess SDF solution with gauze, and applying fluoride varnish to the SDF-treated lesion.
CONCLUSIONS
Although the SDF application protocol is simple and straightforward, the published protocols could be different. Most publications suggested an SDF application time of 60 seconds, which can be long, particularly for young children and older adults.
Topics: Aged; Cariostatic Agents; Child; Child, Preschool; Dental Caries; Fluorides, Topical; Humans; Petrolatum; Quaternary Ammonium Compounds; Silver Compounds
PubMed: 35843730
DOI: 10.1016/j.identj.2022.06.006 -
Current Protocols Jul 2023Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by...
Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents.
Topics: Mice; Humans; Animals; Norovirus; Antiviral Agents; Caliciviridae; Genome; Mammals
PubMed: 37478303
DOI: 10.1002/cpz1.828 -
Autoimmunity Reviews Aug 2022Mucous Membrane Pemphigoid (MMP) is a potentially fatal mucocutaneous autoimmune blistering disease. Autoantibodies are produced against various components of the... (Review)
Review
Mucous Membrane Pemphigoid (MMP) is a potentially fatal mucocutaneous autoimmune blistering disease. Autoantibodies are produced against various components of the dermo-epidermal or mucosal-submucosal junction are referred to as basement membrane zone (BMZ). The hallmark is deposition of of Ig and C3 on the perilesional tissues and in some patients detection of anti-BMZ autoantibodies. A unique characteristic of MMP is that as the blisters or erosions heal, they leave irreversible scarring. This scarring results in serious and catastrophic sequelae that affect the quality of life. Conventional therapy consists of anti-inflammatory and immunosuppressive agents (ISA). In patients who fail conventional therapy or develop significant side effects to them, rituximab (RTX) has been used off label. In this review, the clinical outcomes of patients with MMP treated with RTX were studied. 124 patients were identified, 47.58% being male. 72 patients were treated by the Lymphoma Protocol and 51 by Rheumatoid Arthritis (RA) protocol. Follow up for the entire cohort was 36 months (range 0.5-72). On follow-up 64 patients (51.61%) achieved complete clinical remission (CR) off therapy, 25 patients (20.16%) were in CR on therapy, 5 patients (4.03%) were non-responders, and 9 patients (7.25%) were failures. 52 patients (41.93%) experienced a relapse, after 36 months follow-up. Duration between last RTX infusion and relapse was 10.5 months (range 1-30). Most patients with relapses were treated with additional RTX. A statistically significant better outcome was observed in patients treated with RTX as monotherapy compared to those who received RTX with ISA. Clinical outcomes in patients treated with Lymphoma protocol were better than RA protocol at a statistically significant level. Data on CD20+ B cell depletion and repopulation was limited. Interestingly relapses were seen in patients with CD20+ B cell depletion and after repopulation. In the final analysis, 89 patients (71.77%) were in complete remission. Data in this review indicated that RTX was a useful agent to treat MMP. While a randomized control trial may not be practically possible, better and disease specific protocols need to be developed. When publishing, authors should attempt to provide complete and detailed information. In doing so, they will benefit their colleagues and the patients with MMP they treat with RTX.
Topics: Antigens, CD20; Arthritis, Rheumatoid; Autoantibodies; Autoimmune Diseases; Cicatrix; Female; Humans; Immunosuppressive Agents; Male; Mucous Membrane; Pemphigoid, Benign Mucous Membrane; Pemphigoid, Bullous; Quality of Life; Randomized Controlled Trials as Topic; Recurrence; Retrospective Studies; Rituximab; Treatment Outcome
PubMed: 35688385
DOI: 10.1016/j.autrev.2022.103119 -
Journal of Clinical Pharmacy and... May 2022Betalactam antibiotics are the most frequent cause of hypersensitivity reactions. Rapid drug desensitization (RDD) is a technique that induces temporary tolerance to a...
WHAT IS KNOWN AND OBJECTIVE
Betalactam antibiotics are the most frequent cause of hypersensitivity reactions. Rapid drug desensitization (RDD) is a technique that induces temporary tolerance to a drug allowing a patient to receive the optimal agent. The increased use of RDD and the lack of standardization among available protocols in terms of formulation, starting dose, number of steps and dosing frequency make it essential to determine the safety and appropriate management of these protocols, especially regarding reconstitution, diluents, stability and drug administration in order to guarantee reproducibility. We reviewed betalactam desensitization protocols in a tertiary hospital, in accordance with currently published practices and evaluated its use on patients over a period of three years.
METHODS
(a) We performed a literature search in PubMed, MEDLINE and Google Scholar databases for case reports and/or systematic reviews describing desensitization protocols for betalactam antibiotics. Pharmacokinetic parameters and physicochemical stability were checked for each antibiotic. (b) We retrospectively reviewed inpatients undergoing our antibiotic desensitization protocols from February 2018 to January 2021. Data and outcomes of desensitization procedures were analysed.
RESULTS
We developed nine RDD protocols: meropenem, ceftriaxone, ceftazidime, ampicillin, ceftolozane/tazobactam, cloxacillin, piperacillin/tazobactam, amoxicillin/clavulanate and penicillin G sodium. Five antibiotics have RDD protocols for two different doses, adjusted to patients with impaired renal function. Detailed data (diluent, total dose, volume, concentrations, duration and stability) of the protocol of each antibiotic used are provided. 28 desensitizations were performed in 17 patients, three of them with confirmed allergies by skin test. 26 out of 28 (92.9%) of them were successfully completed, including those three with positive skin results. The pathogens most frequently involved were E. faecalis and P. aeruginosa; both frequently associated with bacterial resistance. Meropenem, ceftriaxone and ceftazidime were the antibiotics most desensitized. 25 out of 26 (96.1%) procedures were successful in resolving the infection.
WHAT IS NEW AND CONCLUSIONS
Detailed information about compounding, dilution and stability is crucial to ensure safe and successful desensitization processes, as well as good coordination between the Allergy and Pharmacy departments. The increase in bacterial resistance to many of the commercially available antibiotics limits the therapeutic options for treating multidrug-resistant infections; in those situations, antibiotic desensitization may be a key therapeutic option. Although there is a broad consensus in limiting the use of RDD to patients with confirmed allergy, in usual clinical practice its application in those strongly suspected of having type I hypersensitivity is still observed. Our betalactam desensitization protocols have shown themselves to be safe and effective, as evidenced by data from the 17 patients on whom they have been tested.
Topics: Anti-Bacterial Agents; Ceftazidime; Ceftriaxone; Drug Hypersensitivity; Humans; Meropenem; Reproducibility of Results; Retrospective Studies; Review Literature as Topic; Tazobactam
PubMed: 34820864
DOI: 10.1111/jcpt.13578