Dr. Stein teaches problem-based clinical reasoning from a clinical case twice a year at the Department of General Medicine, Juntendo University Faculty of Medicine. Problem-based clinical reasoning is to organize clinical information such as medical history, physical examination and laboratory data, into practical problem lists and develop assessment for diagnosis and treatment. Here is the report of the recent case conference to learn problem-based clinical reasoning.
A 36-year-old man admitted to our hospital because of fever, erythema and migratory polyarticular pain. In this case, the listed problems by Dr. Stein were as follows: fever, macular erythema on upper and lower extremities, arthritis of both feet, bilateral Achilles’ tendon pains and enthesitis of ankle joints and Achilles tendons. Based on this problem list, reactive arthritis seemed as the most possible diagnosis. By asking the history of present illness repeatedly, he confessed that he had experienced diarrhea about 5 days before other symptoms started. Stool culture was performed and yielded Salmonella typhimurium (O:4, H:I), so we could conclude the final diagnosis as reactive arthritis after Salmonella infection. Dr. Stein pointed out the importance to distinguish arthritis from arthralgia for correct recognition of patient’s illness and the effectiveness to return to the history after listing problems and possible diagnosis.

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Pyroptosis is defined as a caspase-1 dependent cell death and mainly occurs in macrophages and dendritic cells, accompanied with the release of pro-inflammatory cytokines. Moreover, pyroptosis results in the cellular lysis and release of cytosolic contents, which induce the augmentation of inflammatory reactions. Of note, caspase-1 knockout (KO) causes a protective effect on the sepsis models and improves the survival of mice, in which the IL-1β level was totally suppressed. Thus, caspase-1 activation and possibly pyroptosis play a major role in the mortality of sepsis. AMPs (antimicrobial peptides) are functioning in the first line of defense against invading pathogens. Cathelicidin family of AMPs are recognized in many mammalian species, and LL-37 is the only one human cathelicidin. Besides its broad spectrum of antimicrobial actions, LL-37 has a role in innate immune defense, such as regulation of inflammatory responses and wound healing. Interestingly, LL-37 displays the protective effect on the endotoxemia models. In this review, we introduce a newly identified inhibitory effect of LL-37 on macrophage pyroptosis in vitro and in vivo.

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Cathelicidins are a family of antimicrobial peptides that are found in several mammalian species.They consist of a highly conserved N-terminal prosequences (a cathelin-like domain) and variable C-terminal sequences that is retained within the mature antibacterial peptide. LL-37 is the sole mature cathelicidin peptide found in humans. It is composed of 37 amino acids and exhibits an α-helical structure. Although LL-37 exerts a broad spectrum of antimicrobial activities against bacteria, fungi, and certain viruses, there is currently much focus on the various effects of the peptide on host cells. Growing evidence suggests that LL-37 binds to host cell surface receptors and induces immunomodulatory responses in neutrophils, monocytes/macrophages, dendritic cells, T cells, mast cells, and epithelial cells. LL-37 also acts on endothelial cells and induces cell proliferation, angiogenesis, upregulated adhesion molecule expression, and increased cell stiffness. Moreover, LL-37 is incorporated into host cells and modulates host cell responses by itself or by forming a complex with host/pathogen-derived components. In this review, we describe the effects of LL-37 on endothelial cells and present the findings of our recent study, which revealed that LL-37 contributes to lipopolysaccharide (LPS) clearance by liver sinusoidal endothelial cells by forming complexes with Gram-negative LPS. Finally, we discuss the involvement of LL-37 in atherosclerosis and regeneration.

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Antimicrobial peptides (AMPs) are produced by multicellular organisms and play an important role in innate immunity. AMPs are considered to be promising candidates for novel antibiotics because the emergence of drug-resistant bacteria has become a serious global health problem. There are many advantages of AMPs over conventional antibiotics, including a broad antibacterial spectrum and unique antibacterial mechanisms. Importantly, microbial resistance to membrane-disruptive AMPs is very unlikely to occur rapidly because changes in the target cell membrane cannot occur within a short period. However, antigenicity, cytotoxicity, stability, and production cost are the main difficulties with the application of AMPs for therapeutic purposes. Therefore, modifications of the original AMPs have been performed to overcome these difficulties.
Here we describe our work on the potential therapeutic applications of AMPs as well as the development of beetle defensin-derived AMPs and their multiple functions and applications. The defensin-derived AMPs disrupt negatively charged phospholipids on the cell membrane, showing direct cytotoxic activity against bacteria, fungi, protozoa, and cancer cell lines as well as induce apoptosis by disrupting the mitochondrial membrane; however, it does not demonstrate cytotoxic activity against normal mammalian cells. AMPs also show telomerase inhibition activity. They showed therapeutic effects in MRSA and Escherichia coli (E. coli)-infected mice, with very weak antigenicity. The AMP-immobilized fibers exhibited potent antibacterial activity against Staphylococcus aureus (S. aureus), including MRSA, and the activity was maintained even after repetitive washing and sterilization by autoclaving. These results suggest that the AMP-immobilized fibers are promising for use as novel antimicrobial materials.

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In addition to functioning as a physical barrier, the skin has evolved several innate defense mechanisms for the rapid recognition of and protection against harmful microorganisms. As a part of this process, the skin releases a vast and powerful arsenal of antimicrobial peptides/proteins (AMPs), also called host defense peptides/proteins (HDPs), which are key players in the cutaneous immune system. Although originally named antimicrobial peptides, recent evidence has demonstrated that AMPs/HDPs play additional roles in orchestrating the adaptive immune response, such as the regulation of inflammation, induction of cell proliferation and differentiation, regulation of cytokine/chemokine production, facilitation of cell migration, promotion of wound healing and regulation of tight junction barriers. Additionally, numerous skin diseases show altered expression of AMPs/HDPs in the lesional skin, suggesting the crucial roles of these molecules in the pathophysiology of skin diseases. The purpose of this review is to describe the current knowledge regarding some of the most common and well-known AMPs/HDPs derived from the skin, to discuss the regulation of their expression and their antimicrobial and immunomodulatory functions, and to outline their roles in various skin diseases. We believe that understanding the basic knowledge of skin-derived AMPs/HDPs would provide new insight into the pathophysiology of skin disorders and offer novel therapeutic opportunities for skin infectious diseases.

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The Limulus amoebocyte lysate (LAL) test is the most sensitive and reliable assay for the detection of trace amounts of bacterial endotoxins (lipopolysaccharides, or LPS), and is an accepted in vitro alternative to the rabbit pyrogen test for evaluations of parenteral drugs, biological products, and medical devices. There are three principal LAL tests, which can be categorized as both semi-quantitative and quantitative methods, including gel-clot, turbidimetric, and chromogenic assays. Since the 1970s, these tests have been successfully formulated and commercialized by US and Japanese manufacturers. More recently, in addition to the recombinant factor C-based assay, a novel product containing all of the recombinant coagulation factors from horseshoe crab has been developed, which may lead to the creation of a next generation LAL alternative. Furthermore, there are antimicrobial peptides called “host defense peptides (HDPs)” that play a key role in innate immune responses. The LAL test for HDP-related studies is challenging, because the active site of endotoxin could be masked by the binding with HDPs. Thus, it is very important to properly evaluate the actions of HDPs (human defensins and cathelicidin peptide LL-37) such as the neutralization of LPS, immunostimulatory functions, and anti-endotoxin activity. Moreover, sensitive detection of LPS in cell culture media should be conducted to address the problem of endotoxin contamination in the media. Here, we discuss the progress of LAL-based endotoxin assay technologies, as well as their applications and limitations, with a focus on innovative functional studies of HDPs.

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Objective: A bilateral-bidirectional Glenn procedure is generally performed in patients with a functional single ventricle and a bilateral superior vena cava. In bilateral superior vena cava, unbalanced blood flow due to its unique anatomy and reduced volume due to its small aperture can cause blood stasis, unbalanced pulmonary blood flow, and thrombosis formation. Unifocalization of bilateral superior vena cava, a new surgical technique which makes pulmonary blood flow more equally distributed, was performed and evaluated.
Methods: We retrospectively reviewed the clinical and surgical records of 65 patients who underwent Glenn procedure at the Juntendo University Hospital, Tokyo, from January 1997 to March 2014. Sixteen patients had bilateral superior vena cava anatomy. Unifocalization of superior vena cava was performed in 8 cases and conventional surgery in 8. Perioperative data were evaluated to compare outcomes and clinical courses between the two groups.
Results: There were no significant differences between the 2 groups in age (group 1: 1.0±0.5, group 2: 1.2±1.1, years of age), body weight (group 1: 7.7±2.3, group 2: 6.5±4.3, kg), change in central venous pressure before and after the operation (group 1: 6.5±3.1, group 2: 9.9±6.2, mmHg), postoperative oxygen saturation (group 1: 82±3.3, group 2: 83±9.3, %), duration of surgery (group 1: 371±120, group 2: 439±168, min), or cardiopulmonary bypass time (group 1: 143±38, group 2: 131±53, min). Unilateral blood flow, which is purportedly better than bilateral bidirectional Glenn procedure, was achieved without any disadvantages that are reported of the conventional procedure.
Conclusion: There was no distinct advantage or disadvantage to using the new method.

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Objective: To prevent muscle weakness and atrophy that are caused by discontinuing sports activities, we investigated the effect of local cooling and blood flow restriction (BFR) during detraining.
Methods: Eleven healthy men (23.8±2.1 years) performed elbow flexion resistance training three times per week for 6 weeks. After training, the subjects were instructed to limit the upper arm activity within their activities of daily living level during 3 weeks of detraining. During the detraining period, one arm was used as a control (CON, n=11); the other arm was used under the condition of cooling at medial side of upper arm by an ice bag (ICE, n=6), or under BFR (BFR, n=5). Measurements included elbow flexion torque at angular speeds of 60°/s and 120°/s under concentric contraction and isometric contraction (IM) and cross-sectional area of the upper arm.
Measurements were conducted at pre-training (Pre); post-training (Post); and after 1week (W1), 2 weeks (W2) and 3 weeks (W3) of detraining.
Results: IM torque and cross-sectional area significantly increased following training in all conditions. During detraining, IM torque significantly decreased under the CON condition (Post, 73.2±19.9 Nm; W3, 64.3±11.6 Nm), but no significant changes were observed under ICE condition. However, BFR condition significantly increased following detraining. The percent change in each condition during detraining was significant between the CON and BFR conditions at W2 and W3. Cross-sectional area significantly decreased following detraining in all conditions.
Conclusions: Local cooling and BFR suppressed muscle weakness that was caused by detraining.

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Objective: The aim of this study was to investigate the associations of anti-Müllerian hormone (AMH) levels with physique and 25-hydroxyvitamin D (25OH-D) levels in healthy women of reproductive age based on measurements of nutritional status and physical constitution.
Materials and Methods: Subjects comprised 108 non-obese women (age range, 21-39 years) who underwent examination of their physique, blood biochemistry and nutritional state. For data analysis, subjects were first divided by age. AMH levels were grouped by serum 25OH-D concentration using Holick’s classification: deficiency, <30 ng/ml; and sufficiency, ≥30 ng/ml.
Results: Mean levels were 25.2±8.4 ng/ml for serum 25OH-D and 4.9±2.4 ng/ml for AMH. Overall, 76 women (70.4%) were diagnosed with 25OH-D deficiency. Serum AMH levels were significantly lower in subjects with 25OH-D deficiency (4.5±2.5 ng/ml) than in those with 25OH-D sufficiency (5.7±1.9 ng/ml; p<0.01).
Significant differences were seen in the frequency of subjects with 25OH-D deficiency and sufficiency between low AMH (<2.2 ng/ml) status and normal AMH (≥2.2 ng/ml)(16/17 [94.1%] vs. 1/17 [5.9%] for low AMH status; 60/91 [65.9%] vs.31/91 [34.1%] for normal AMH status, respectively; p<0.05). Independent predictors of serum AMH levels≥2.2 ng/ml were serum 25OH-D level (p<0.05) and age (p<0.05) according to binary logistic regression analysis.
Conclusions: Decreased serum AMH level is associated with vitamin D deficiency, but is unrelated to physique state in this population.

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Ten years have passed since the opening of the Juntendo University Nerima Hospital in July 2005. Radiation therapy started at this facility in September 2005 with one linear accelerator (Linac) considered state-of-the-art at the time. Our facility has only this one treatment device, and using this device, we have performed a lot of conventional irradiation as well as high-precision radiation therapy, such as stereotactic irradiation, intensity-modulated radiation therapy, and image-guided radiation therapy. We present the analyzed results of the past 10 years and introduce the patient characteristics of radiation therapy at the Juntendo University Nerima Hospital.

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