A trial of somatic gene targeting in vivo with an adenovirus vector

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ(+ )gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means

Position of meristems and the angles of the cell division plane regulate the uniqueness of lateral organ shape

花びらの形が葉と違う仕組みを解明. 京都大学プレスリリース. 2022-12-12.Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we examined how cell proliferative zones in primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaf organs differ from those of non-treated foliage leaves of Arabidopsis thaliana, with a focus on the accumulation pattern of ANGUSTIFOLIA3 (AN3) protein, a key element for leaf meristem positioning. We found that PATI-induced leaf shape changes were correlated with cell division angle but not with meristem positioning/size or AN3 localisation. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position, due to altered AN3 expression patterns, and different distributions of cell division angles. A numerical simulation showed that meristem position majorly affected the final shape but biased cell division angles had a minor effect. Taken together, these results suggest that the unique shapes of different lateral organs depend on the position of the meristem in the case of floral organs and cell division angles in the case of leaf organs with different auxin flow

Radiation in an emergency situation: attempting to respect the patient's beliefs as reported by a minor

Background　Each individual's unique health-related beliefs can greatly impact the patient-clinician relationship. When there is a conflict between the patient's preferences and recommended medical care, it can create a serious ethical dilemma, especially in an emergency setting, and dramatically alter this important relationship. Case presentation　A 56-year-old man, who remained comatose after out-of-hospital cardiac arrest, was rushed to our hospital. The patient was scheduled for emergency coronary angiography when his adolescent daughter reported that she and her father held sincere beliefs against radiation exposure. We were concerned that she did not fully understand the potential consequences if her father did not receive the recommended treatment. A physician provided her with in depth information regarding the risks and benefits of the treatment. While we did not want to disregard her statement, we opted to save the patient's life due to concerns about the validity of her report. Conclusions　Variations in beliefs regarding medical care force clinicians to incorporate patient beliefs into medical practice. However, an emergency may require a completely different approach. When faced with a patient in a life-threatening condition and unconscious, we should take action to prioritize saving their life, unless we are highly certain about the validity of their advance directives

The Soluble Factor from Oral Cancer Cell Lines Inhibits Interferon-γ Production by OK-432 via the CD40/CD40 Ligand Pathway

OK-432 is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously reported that OK-432 treatment can induce the production of high levels of IFN-γ from peripheral blood mononuclear cells (PBMCs). Moreover, the IFN-γ production from PBMCs by OK-432 is impaired by conditioned media (CM) from oral cancer cells. To determine the inhibitory mechanism of IFN-γ production by CM, the genes involved in IFN-γ production was retrieved by cDNA microarray analysis. We found that CD40 played a key role in IFN-γ production via IL-12 production. Although the expression levels of CD40 were upregulated by OK-432 treatment in PBMCs, CM inhibited OK-432-induced CD40 expression. These findings suggest that uncertain soluble factor(s) in CM may suppress IFN-γ production via the CD40/CD40L–IL-12 axis in PBMCs.(1) Background: OK-432 is a penicillin-killed, lyophilized formulation of a low-toxicity strain (Su) of Streptococcus pyogenes (Group A). It is a potent immunotherapy agent for several types of cancer, including oral cancer. We previously showed that (i) OK-432 treatment induces a high amount of IFN-γ production from peripheral blood mononuclear cells (PBMCs), and (ii) conditioned medium (CM) from oral cancer cells suppresses both the IFN-γ production and cytotoxic activity of PBMCs driven by OK-432. The aim of this study was to determine the inhibitory mechanism of OK-432-induced IFN-γ production from PBMCs by CM. (2) Methods: We performed cDNA microarray analysis, quantitative RT-PCR, and ELISA to reveal the inhibitory mechanism of CM. (3) Results: We found that CD40 plays a key role in IFN-γ production via IL-12 production. Although OK-432 treatment upregulated the expression levels of the IL-12p40, p35, and CD40 genes, CM from oral cancer cells downregulate these genes. The amount of IFN-γ production by OK-432 treatment was decreased by an anti-CD40 neutralizing antibody. (4) Conclusions: Our study suggests that uncertain soluble factor(s) produced from oral cancer cells may inhibit IFN-γ production from PBMCs via suppressing the CD40/CD40L–IL-12 axis

Emotional work stress reactions of emergency medical technicians involved in transporting out-of-hospital cardiac arrest patients with “do not attempt resuscitation” orders

Background Emergency medical technicians (EMTs) may be subjected to emotional stress during patient treatment/transport. In Japan, dispatched EMTs must attempt resuscitation in all cases of out-of-hospital cardiac arrest (OHCA), including patients with “do not attempt resuscitation” (DNAR) orders and patients whose families do not support resuscitation. We described the characteristics, prevalence, and outcomes of OHCA/DNAR patients, and aimed to identify factors associated with EMT stress when treating them. Methods We included OHCA patients transported by EMTs in the city of Okayama from 2015 to 2019. We identified patients with DNAR orders based on emergency medical service (EMS) records, then EMTs completed questionnaires regarding the management of those patients and EMTs’ emotions. Results Among 3079 eligible OHCA patients, 122 patients (4%) had DNAR orders (DNAR group), and 2957 (96%) patients had no DNAR orders (no DNAR group). Based on responses from 243 EMT participants involved in OHCA/DNAR transports, we divided EMTs into high stress (73/243, 30%) and low stress (170/243, 70%) groups. EMTs experienced emotional stress from treating patients with family physician orders to transport (AOR: 4.74, 95% CI: 2.35–9.56) and those for whom prehospital defibrillation was performed (AOR: 20.7, 95% CI: 3.10–137.9). Conclusions Approximately 30% of EMTs providing resuscitation to OHCA/DNAR patients experienced high levels of stress. Establishment of a prehospital emergency system incorporating physician medical direction and updated guidelines for treating patients with DNAR orders may reduce the psychosocial stress of EMTs

Molecular genetic analysis of pyridoxine-nonresponsive homocystinuric siblings with different blood methionine levels during the neonatal period

Two mutations in the cystathionine β-synthase (CBS) gene were found in two Japanese siblings with pyridoxine non-responsive homocystinuria who had different methionine levels in their blood during the neonatal period. Both patients were com-pound heterozygotes of two mutant alleles:one had an A-to-G transition at nucleotide194 (A194G) that caused a histidine-to-arginine substitution at position 65 of the protein (H65R), while the other had a G-to-A transition at nucleotide346 (G346A) which resulted in a glycine-to-arginine substitution at position 116 of the protein (G116R). The two mutant proteins were separately expressed in Escherichia coli, and they completely lacked catalytic activity. Despite their identical genotypes and almost equal protein intake, these siblings showed different levels of blood methionine during the neonatal period, suggesting that the level of methionine in blood is determined not only by the defect in the CBS gene and protein intake, but also by the activity of other enzymes involved in methionine and homocysteine metabolism, especially during the neonatal period. Therefore, high-risk newborns who have siblings with homocystinuria, even if the level of methionine in their blood is normal in a neonatal mass screening, should be followed up and diagnosed by an assay of enzyme activity or a gene analysis so that treatment can be begun as soon as possible to prevent the development of clinical symptoms. In addition, a new, more sensi-tive method for the mass screening of CBS deficiency in neonates should be developed

身体消費エネルギー量と総白血球数，白血球分画の関係

内科学（第三）講座 講座主任　内潟安子教授退任記念特別

The Impact of Medical Students Teaching Basic Life Support to Laypersons

Basic life support (BLS) courses for laypersons, including cardiopulmonary resuscitation (CPR) training, is known to improve outcomes of out-of-hospital cardiac events. We asked medical students to provide BLS training for laypersons as a part of their emergency medicine education and evaluated the effects of training on the BLS skills of laypersons. We also used a questionnaire to determine whether the medical students who provided the BLS training were themselves more confident and motivated to perform BLS compared to students who did not provide BLS training. The proportions of laypersons who reported confidence in checking for a response, performing chest compressions, and automated external defibrillator (AED) use were significantly increased after the BLS training. The proportions of medical students who reported increased confidence/motivation in terms of understanding BLS, checking for a response, chest compression, use of AED, and willingness to perform BLS were significantly greater among medical students who provided BLS instructions compared to those who did not. BLS instruction by medical students was associated with an improvement in laypersons’ CPR accuracy and confidence in responding to cardiac arrest. The results indicate that medical students could gain understanding, confidence, and motivation in regard to their BLS skills by teaching BLS to laypersons

Calciphylaxis as a Catastrophic Complication in a Patient with POEMS Syndrome

Calciphylaxis is a vascular calcification-cutaneous necrosis syndrome, usually seen in patients with end-stage renal disease and secondary hyperparathyroidism. We report a 57-year-old polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome patient complicated with extensive skin ulcers due to calciphylaxis. He first noted a painful cutaneous ulcer on his left thigh, and then skin lesions rapidly worsened, resulting in multiple intractable ulcers with gangrene on his legs and trunk in a few months. Serum vascular endothelial growth factor (VEGF) was markedly elevated. Biopsy samples from his skin ulcers showed the deposition of calcium in the medial layer of cutaneous vessels, this finding being compatible with calciphylaxis. This is the second reported case with POEMS syndrome complicated with calciphylaxis. Both patients had no evidence of renal failure, hyperparathyroidism, or clotting disorders. The pathogenic link between POEMS syndrome and calciphylaxis is still unclear, but VEGF is known to regulate vascular calcification, in cooperation with bone morphogenetic proteins. Further, corticosteroid and several proinflammatory cytokines activate nuclear factor-κB pathway, known as the final common pathway leading to vascular calcification. Taken together, we consider that POEMS syndrome can be an independent risk condition for calciphylaxis