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PubMed1982

[Current immunologic aspects of kidney transplantation].

The results of clinical kidney transplantation are mainly dependent on immunologic factors many of which are unknown or of unspecified importance. Blood transfusions have a favorable effect on graft prognosis, although our knowledge about optimal transfusion protocols and transfusion-induced mechanisms is still incomplete. The value of HLA-typing is controversial: whereas compatibility of the "classical" HLA-A,B,C antigens improves graft survival only moderately, HLA-DR typing, routinely performed for the last 3 years only, might be of greater importance. In addition, non-HLA systems, such as endothelial/monocytic antigens or the Lewis blood group system, appear to play a role in graft rejection. The individual immune reactivity a recipient to a large extent determines the fate of a graft. The multifactorial dependence of graft prognosis is discussed in this report.

Blood Group Incompatibility↗

PubMed1995

No effect of 60 Hz electromagnetic fields on MYC or beta-actin expression in human leukemic cells.

Epidemiological studies have shown weak correlations between exposure to extremely low-frequency electromagnetic fields (ELF EMFs) and the incidence of several cancers, particularly childhood leukemias, although negative studies have also been reported. These observations have prompted a broad range of in vitro cellular studies in which effects of ELF EMFs have been observed. However, no reported response has been replicated widely in independent laboratories. One potentially important response is the rapid activation of proto-oncogenes and other genes in human leukemic (HL60) cells and a wide variety of other eukaryotic cells, because of the role of these genes in cell proliferation. We describe quantitative Northern analysis of MYC and beta-actin mRNAs from HL60 cells exposed to fields under conditions very similar to those reported previously to activate these genes, namely 60 Hz sinusoidal magnetic fields of 0.57, 5.7 or 57 microT for 20 min. In addition we have used a new design of field-exposure system and introduced a number of other modifications to the protocol to optimize any response. We have also developed a novel method providing enhanced accuracy for the quantitative measurement of mRNA. No significant effect of ELF EMFs on gene expression was observed using any of these systems and analytical methods.

Actins↗

PubMed1976

Randomized clinical trial of antithymocyte globulin in cadaver renal allograft recipients: importance of T cell monitoring.

Despite incontrovertable evidence demonstrating the unique immunosuppressive capabilities of antihymocyte globulin (ATG) in animals, its value in clinical transplantation has remained inconclusive. A multicenter, prospective study was undertaken in an attempt to determine this value. Cadaver renal allograft recipients were randomized into two treatment groups: prednisone and azathioprine only, or prednisone, azathioprine, and ATG. Four to 36 month follow-up of our first 50 recipients, supported by results in over 200 patients from other centers, permits the following observations. (1) Allograft survival can be improved by nearly 20 percent in recipients treated with active ATG preparations. (2) Presently available assays performed in vitro and on subhuman primate allograft survival, though capable of excluding inactive preparations, cannot quantitate precisely the immunosuppressive capacity of active batches of ATG. (3) Monitoring circulating T cell levels in patients receiving ATG appears to offer the best means of defining immunosuppressive potency and the variation in response to the agent among identically treated individuals. It is concluded that ATG therapy can be beneficial in renal transplantation; but monitoring of recipients' T cell level is desirable during therapy to determine the dosage required. Furthermore, the optimal immunosuppressive protocol can be approximated by modifying the regimen according to individual variations in response, thus limiting the infectious complications of excessive immunosuppression.

Adolescent↗

PubMed2025

Impact of RNA extraction on respiratory microbiome analysis using third-generation sequencing.

BACKGROUND: The respiratory microbiome, which comprises bacteria, fungi, and viruses, plays a crucial role in respiratory health and disease. However, its study is limited by the low microbial biomass in respiratory samples and the dominance of host RNA. Metatranscriptomics offers comprehensive insights into active microbial communities and their interactions with the host but requires optimized RNA extraction protocols for robust and unbiased analysis. This study evaluated two RNA extraction kits—one employing chemical lysis (CL) and another combining chemical and mechanical lysis (CML)—to determine their effectiveness for metatranscriptomic analysis of respiratory samples. RESULTS: The CML protocol significantly increased double-stranded DNA (dsDNA) library yields, leading to higher sequencing read counts for both sample types (p < 0.0001). The read length was unaffected by the lysis protocol for the BAL and NPS samples. Taxonomic profiling revealed that CML enhanced the detection of robust microorganisms, such as gram-positive bacteria and fungi, without compromising viral detection. CONCLUSIONS: The CML protocol demonstrated superior recovery of genetic material, particularly for fungi and gram-positive bacteria, making it better suited for comprehensive metatranscriptomic analyses. These findings underscore the need for tailored RNA extraction strategies on the basis of sample type and research objectives. Optimized metatranscriptomic protocols are pivotal for advancing our understanding of the respiratory microbiome and its role in health and disease.

Microbiota↗

PubMed1978

Mathematical optimization of glaucoma visual field screening protocols.

There is potential for significantly shortening the time required for visual field screening protocols by a precise specification of the number, exact location, and sequence of points to be tested. Through statistical and mathematical methods, protocols have been developed for maximizing the probability of detecting at least one visual field defect in a subject who is a risk for early glaucomatous field loss. The mathematical formulation was derived in a generalized manner so that it could be applied to most kinetically or statically determined visual field screening methods.

Glaucoma↗

PubMed1992

Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction.

In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.

Agrobacterium tumefaciens↗

PubMed1995

[Adequate neuropsychological evaluation of dementia].

The authors discuss the protocol of the Optimal Neuropsychological Evaluation of Dementias of Montreal and its possible application in Brazil. This protocol is important for evaluation in neuropsychological evolution of dementia of the Alzheimer type and for bringing to the fore distinct affected cognitive profiles as much on a transversal base as longitudinal. The authors believe that its application would contribute to the progress of research in neurology, as in the clear distinction between normal aging and pathological aging.

Alzheimer Disease↗

PubMed1992

Multidisciplinary protocol for determining aminoglycoside dosage.

A protocol for determining optimal dosages of aminoglycosides early in therapy is described, and the effectiveness of the protocol is evaluated. The protocol was developed jointly by physicians and pharmacists at a 550-bed hospital to ensure that surgical patients prescribed aminoglycosides were quickly and consistently put on a safe and effective course of therapy. Physicians select an aminoglycoside and calculate a loading dose and initial maintenance dosage by using a nomogram printed on an antimicrobial order form. Nurses are trained to administer and document aminoglycoside doses accurately and to draw blood samples at the correct times. Pharmacists order serum aminoglycoside concentration assays, analyze the results, and recommend changes in dosage when necessary. To evaluate the effectiveness of the dosing protocol, the records of surgical patients treated before and after the protocol was in place were reviewed. Compared with the control group, a higher percentage of patients treated under the protocol were receiving therapeutic, nontoxic dosages of aminoglycosides within 48 hours of the start of therapy. In addition, fewer serum drug concentration tests were ordered per patient under the protocol, and the percentage of concentration determinations useful for analysis was higher. The mean duration of aminoglycoside therapy was identical before and after the protocol was instituted, and nephrotoxic reactions tended to be less common among the protocol patients. An aminoglycoside dosing protocol requiring the cooperation of pharmacists, nurses, and physicians provides a consistent, safe, and effective means of managing aminoglycoside therapy for the hospitalized patient.

Adult↗

PubMed1986

Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies.

This paper describes an experimental protocol designed to optimize 13C NMR spectra from the liver of the living anesthetized rat at 1.9 T. The protocol involves the use of a Helmholtz NMR coil which is positioned around the liver after surgical exposure. 1H decoupling is facilitated by double tuning this coil to both the 1H and the 13C frequencies. The protocol was shown to be suitable for studying the hepatic metabolism of 13C-labeled substrates in vivo by investigating the metabolism of [3-13C]alanine. Labeled glucose, glutamate, glutamine, and aspartate were formed and detected by 13C NMR in vivo in this experiment. The labeling patterns in these metabolites provided evidence that the major flow of alanine carbon into the Krebs cycle is via the pyruvate carboxylase reaction rather than through pyruvate dehydrogenase.

Alanine↗

PubMed1992

Development of immunotherapeutic strategies for the treatment of malignant neoplasms.

Focused preclinical studies have been used to gain insight into the mechanism of therapeutic activity of cytokines, growth factors and biological response modifiers (BRMs). These data can then be used to develop a clinical hypothesis to facilitate the development of these new biological drugs. In this manuscript, we discuss a number of preclinical and clinical studies using interferon-gamma, IL-2, and the colony stimulating factors. The importance of the systematic profiling of the biological activity of such biological drugs is emphasized and we discuss the utility of the mechanistic data in their clinical development. The overall preclinical approach identifies the cellular, biochemical or gene regulatory event that is associated with the therapeutic activity of a biologic and this surrogate (be it biological, chemical, or quality of life) is then used to optimize the clinical protocol in a phase 1b trial. This, in theory, results in the rapid identification of the optimal dose, schedule and route of administration for subsequent testing in a phase II/III clinical trial.

Animals↗

PubMed1994

A study of simulated annealing protocols for use with molecular dynamics in protein structure prediction.

The method of simulated annealing can be of use in protein structure prediction by homology modelling where side chain conformations must be predicted. In this study an attempt has been made to optimize a molecular dynamics method for this purpose. Heating and cooling protocols to maximize the accuracy of the predictions have been developed. The optimized protocol involves cooling from 3000 to 0 K over 20 ps while simultaneously introducing the non-bonded energy term. The use of a 'soft' non-bonded interaction energy term in place of a standard 6-12 potential is found to be important. The reliability of the predictions has been analysed in terms of the environment of the residues (solvent accessibility) and the degree of uncertainty in the structure (number of unknown torsion angles). Depending on these factors the percentage of unknown side chain torsion angles that are correctly predicted within 30 degrees ranges from approximately 50 to 75%. Potential problems and limitations of the method are discussed.

Amino Acid Sequence↗

PubMedDate not supplied

High-throughput DNA preparation system.

A system demonstrating the feasibility of high-throughput, centrifugation-based DNA separations and purifications has been constructed and tested. Samples are currently processed at a rate of 96 in approximately 2-3 h. The device implements an automation-optimized alkaline lysis protocol for the rapid extraction of plasmid or cosmid DNA from 1-ml bacteria cultures. The conditions for optimal culturing in deep-well (96 x 1 ml) microwell plates have been developed, and all sample manipulations are done within these plates. The use of microwell plates was essential to obtain high throughput and make manipulations following the DNA preparation (prep) easier because they can then be manipulated using a variety of commercially available robots. The entire prep system is constructed above a Beckman GPR centrifuge and operated under Macintosh IIcx control. This device has systems for fluid handling, microwell-plate manipulations, and centrifuge rotor alignment.

Automation↗

PubMed1988

The impact of aggressive debulking surgery and cisplatin-based chemotherapy on progression-free survival in stage III and IV ovarian carcinoma.

Forty consecutive patients with stage III and IV invasive ovarian carcinoma were treated on a phase II protocol consisting of optimal debulking surgery, induction cisplatin, cisplatin, doxorubicin, and cyclophosphamide (PAC) chemotherapy, 6-month interval laparoscopy, reinduction cisplatin, PAC chemotherapy, and second-look procedure. All 40 patients have either disease progression or have completed the 12-month protocol. Eighty-seven percent of the patients (35) underwent optimal (less than or equal to 2 cm residual) debulking surgery before chemotherapy, in spite of the fact that 50% (20) were referred to Roswell Park Memorial Institute (RPMI) as inoperable after initial surgery elsewhere. There were no postoperative deaths and chemotherapy was started in less than or equal to 14 days in 97% of the patients. Of the 40 patients, 30% (12) achieved a pathologic complete remission (11) or a clinical complete remission (one patient refused second-look surgery). The estimated 3-year survival rate was 62%, but the 3-year progression-free survival rate was only 29%. The median survival time was 48 months. The estimated 3-year progression-free survival rate was 31% for residual disease less than or equal to 2 cm. For the five patients with residual disease greater than 2 cm, four died within 3 years. The median survival time of patients with less than or equal to 2 cm residual disease was 48 months, as compared with 21 months for those with greater than 2 cm residual disease. Although the estimated 3-year survival rate of 62% is noteworthy, the 3-year progression-free survival rate of only 29% is probably indicative that in spite of extensive debulking surgery and cisplatin-based chemotherapy as used in this protocol, the long range proportion of patients "cured" will remain small.

Adenocarcinoma↗

PubMed2026

Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.

Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000 bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.

Polymerase Chain Reaction↗

PubMed1987

Cancer therapy by biological response modifiers.

Biological response modifiers (BRMs) are agents or approaches that modify the relationship between the tumor and host by modifying the host's biological responses to tumor cells, with resultant therapeutic effects. BRMs include immunomodulators and components of the immune system, e.g. cytokines, antibodies and effector cells. A central challenge in the development of BRMs for therapy of cancer patients is to determine the main mechanism of action of each agent and by monitoring for effects of treatment on the key parameters, optimize the treatment protocol. The experience to date with interferons and with interleukin-2 (IL-2) is summarized, to illustrate both the promise of BRMs and the difficulties in determining the optimal therapeutic regimen and in turn this potential for more effective treatment of cancer. A new sequence of clinical trials for clinical evaluation of BRMs is described, with an emphasis on treatment of patients with minimal tumor burden.

Animals↗

PubMed2025

A xeno-free protocol for rapid differentiation of human iPSC-derived microglia from the KOLF2.1J reference line.

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Adapted from Dräger et al. (1), our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in disease modeling, neuroinflammation research, and therapeutic screening.

Journal Article↗

PubMed1989

Synthesis and characterization of mu-conotoxin IIIa.

mu-Conotoxin IIIa, a voltage-dependent sodium channel neurotoxin, has been synthesised using solid-phase peptide synthesis employing 9-fluorenylmethoxycarbonyl chemistry. After cleavage from the resin, the peptide was isolated by reverse-phase HPLC and then the six acetamidomethyl groups were removed by treatment with mercuric acetate. The reduced product so formed was purified by reverse-phase HPLC. Protocols were developed to optimize the oxidation of the cysteine residues to form disulphide bonds. Protocols employed using air oxidation together with 2-mercaptoethanol were the most effective. As complete oxidation was never obtained the oxidised peptide was purified by reverse-phase HPLC. The activity of our products was monitored using [3H]saxitoxin binding to eel membranes. The oxidised product was able to completely block [3H]saxitoxin binding in a competitive manner. Lineweaver-Burke analysis of [3H]saxitoxin binding gave a Ki of 1.5 nM, IC50 was determined as 26.6 nM. It was also shown that the pure synthetic mu-conotoxin IIIa had the same retention time on reversephase HPLC as the natural conotoxin IIIa. Thus an active toxin has been synthesised that can be used to probe sodium channels.

Binding Sites↗

PubMed1990

A general method of optimizing automated DNA synthesis to decrease chemical consumption to less than half.

An automated DNA synthesis protocol (FC3) has been optimized to decrease the cost of chemical consumption by two- to threefold. Anion-exchange chromatography at pH 12.5 in a Mono Q (Pharmacia) column was used to analyze the oligonucleotides we synthesized. From these analyses, we formulated the FC3 program and a procedure by which a DNA synthesizer designer or operator can use 18-base-long homopolymers of A, G, C, and T to optimize the reaction times, reagent concentrations, solvent wash conditions, and the many steps in the synthesis cycle.

Automation↗