Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants.

BACKGROUND: Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage. METHODS: Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-ß were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared. RESULTS: Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-ß was associated with higher migratory activity of the superficial-zone chondrocytes. CONCLUSIONS: In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-ß combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Safety and immunogenicity of the Pfizer/BioNTech SARS-CoV-2 mRNA third booster vaccine dose against the BA.1 and BA.2 Omicron variants.

BACKGROUND: The Omicron variant of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) was identified in Japan in November 2021. This variant contains up to 36 mutations in the spike protein, the target of neutralizing antibodies, and can escape vaccine-induced immunity. A booster vaccination campaign began with healthcare workers and high-risk groups. The safety and immunogenicity of the three-dose vaccination against Omicron remain unknown. METHODS: A total of 272 healthcare workers were initially evaluated for long-term vaccine safety and immunogenicity. We further established a vaccinee panel to evaluate the safety and immunogenicity against variants of concern (VOCs), including the Omicron variants, using a live virus microneutralization assay. FINDINGS: Two-dose vaccination induced robust anti-spike antibodies and neutralization titers (NTs) against the ancestral strain WK-521, whereas NTs against VOCs were significantly lower. Within 93-247 days of the second vaccine dose, NTs against Omicron were completely abolished in up to 80% of individuals in the vaccinee panel. Booster dose induced a robust increase in anti-spike antibodies and NTs against the WK-521, Delta, and Omicron variants. There were no significant differences in the neutralization ability of sera from boosted individuals among the Omicron subvariants BA.1, BA.1.1, and BA.2. Boosting increased the breadth of humoral immunity and cross-reactivity with Omicron without changes in cytokine signatures and adverse event rate. CONCLUSIONS: The third vaccination dose is safe and increases neutralization against Omicron variants. FUNDING: This study was supported by grants from AMED (grants JP21fk0108104 and JP21mk0102146).

The Contribution of Deleterious Rare Alleles in ENPP1 and Osteomalacia Causative Genes to Atypical Femoral Fracture.

CONTEXT: Atypical femoral fractures (AFFs) are very rare atraumatic or mild trauma fractures in the subtrochanteric region or femoral shaft. Some unique genetic variants in Asian populations might confer susceptibility to AFF, since the incidence of AFFs is higher in Asian populations. OBJECTIVE: Because rare variants have been found to be causative in some diseases and the roles of osteomalacia causative genes have not been reported, we investigated rare variants in genes causing abnormal mineralization. METHODS: Exome sequencing was performed to detect variants in gene coding and boundary regions, and the frequencies of deleterious rare alleles were compared between Japanese patients with AFF (n = 42) and controls of the 4.7KJPN panel of Tohoku Medical Megabank by whole genome sequencing (n = 4773). RESULTS: The frequency of the deleterious rare allele of ENPP1 was significantly increased in AFF (P = .0012, corrected P [Pc] = .0155, OR 4.73, 95% CI 2.15-10.40). In multigene panel analysis, the frequencies of deleterious rare alleles of candidate genes were increased in AFF (P = .0025, OR 2.72, 95% CI 1.49-4.93). Principal component analysis of bone metabolism markers identified a subgroup of patients with AFF with higher frequencies of deleterious rare alleles in ENPP1 (P = 4.69 × 10-5, Pc = .0006, OR 8.47, 95% CI 3.76-19.09) and the candidate genes (P = 1.08 × 10-5, OR 5.21, 95% CI 2.76-9.86). CONCLUSION: AFF is associated with genes including ENPP1 that cause abnormal mineralization, suggesting that osteomalacia is an underlying condition predisposing to AFF and that higher incident rates of AFFs in Asian populations might be explained by the genetic risk factors including ENPP1.

Photodynamic therapy using intra-articular Photofrin for murine MRSA arthritis: biphasic light dose response for neutrophil-mediated antibacterial effect.

BACKGROUND AND OBJECTIVE: Bacterial arthritis does not respond well to antibiotics and moreover multidrug resistance is spreading. We previously tested photodynamic therapy (PDT) mediated by systemic Photofrin® in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) arthritis, but found that neutrophils were killed by PDT and therefore the infection was potentiated. STUDY DESIGN/MATERIALS AND METHODS: The present study used an intra-articular injection of Photofrin® and optimized the light dosimetry in order to maximize bacterial killing and minimize killing of host neutrophils. MRSA (5 × 10(7) CFU) was injected into the mouse knee followed 3 days later by 1 µg of Photofrin® and 635-nm diode laser illumination with a range of fluences within 5 minutes. Synovial fluid was sampled 6 hours or 1-3, 5, and 7 days after PDT to determine MRSA colony-forming units (CFU), neutrophil numbers, and levels of cytokines. RESULTS: A biphasic light dose response was observed with the greatest reduction of MRSA CFU seen with a fluence of 20 J cm(-2), whereas lower antibacterial efficacy was observed with fluences that were either lower or higher. Consistent with these results, a significantly higher concentration of macrophage inflammatory protein-2, a CXC chemokine, and greater accumulation of neutrophils were seen in the infected knee joint after PDT with a fluence of 20 J cm(-2) compared to fluences of 5 or 70 J cm(-2). CONCLUSION: PDT for murine MRSA arthritis requires appropriate light dosimetry to simultaneously maximize bacterial killing and neutrophil accumulation into the infected site, while too little light does not kill sufficient bacteria and too much light kills neutrophils and damages host tissue as well as bacteria and allows bacteria to grow unimpeded by host defense.

CD4(+)CD8(+) thymocytes are induced to cell death by a small dose of puromycin via ER stress.

When the CD4(+)CD8(+) thymic lymphoma cells were treated with puromycin, we found that most of the cells died at 0.3-1 microg/ml of puromycin within 24h. However, cell death was greatly reduced when the dose of puromycin was increased. Similar dose-pattern of cell death was observed in thymocytes and the sensitivity to puromycin was greater in CD4(+)CD8(+) thymocytes than CD4(+)CD8(-) thymocytes. The induction of apoptosis was blocked by the protein synthesis inhibitor cycloheximide, and to some extent by transfection of Bcl-xL or Bcl-2 genes. Expression of GRP78 was up-regulated after treatment with a small dose of puromycin, and the cell death by puromycin was blocked in the presence of caspase 12 inhibitor. These results indicated that the induction of cell death by low-dose puromycin was due to endoplasmic reticulum stress. Furthermore, we found that dexamethasone, a synthetic glucocorticoid, and puromycin worked synergistically to induce cell death in thymocytes.

Cementless total hip replacement: past, present, and future.

Cementless total hip replacement (THR) is rapidly being accepted as the surgery for arthritic diseases of the hip joint. The bone-ingrowth rate in porous-type cementless implants was about 90% over 10 years after surgery, showing that biological fixation of cementless THR was well maintained on both the stem and cup sides. As for the stress shielding of the femur operated using a distal fixation-type stem, severe bone resorption was observed. The severe bone resorption group showed continuous progression for more than 10 years after surgery. Stem loosening directly caused by stress shielding has been considered less likely; however, close attention should be paid to bone resorption-associated disorders including femoral fracture. Cementless cups have several specific problems. It is difficult to decide whether a cup should be placed in the physiological position for the case of acetabular dysplasia by bone grafting or at a relatively higher position without bone grafting. The bone-ingrowth rate was lower in the group with en bloc bone grafting, and the reactive line was frequently noted in the bone-grafted region. Although no data indicated that en bloc bone grafting directly led to poor outcomes, such as loosening, cup placement at a higher site without bone grafting is now selected by most operators. The polyethylene liner in a cementless cup is thinned due to the metal cup thickness; however, it has been suggested that the apparent relation between the cup size and the wear rate was absent as long as a cementless cup is used. Comparative study indicated cementless THR was inferior with regard to the yearly polyethylene wear rate and incidence of osteolysis on both the stem and cup sides. Meta-analysis study on the survival rate between cement and cementless THR reported that cemented THR was slightly superior. It should be considered that specific problems for cementless THR, especially with regard to polyethylene wear, do occur.

Epidemiological patterns of traumatic musculoskeletal injuries and non-traumatic disorders in Japan Self-Defense Forces.

BACKGROUND: The epidemiological patterns of musculoskeletal injuries or disorders in military personnel have not been well documented and a better understanding is required for proper preventative measures and treatment. Here, we investigated musculoskeletal injuries or disorders among members of the Japan Self-Defense Forces. METHODS: All orthopedic patients (n = 22,340) who consulted to Japan Self-Defense Forces Hospitals were investigated for their type of injury or disorder, the injured body part, the mechanism, and the cause of injuries. RESULTS: Thirty-nine percent of the cases were classified as traumatic injuries, and 61% were classified as non-traumatic disorders. Of the traumatic injury patients, the injured body part was the upper extremity in 32%, the trunk in 23%, and the lower extremities in 45% of the cases. The most common injured body location was the knee followed by the hand/finger and ankle. Exercise was the most common cause of injury, followed by traffic accident and military training. Contusions were the most common traumatic injuries, followed by sprains and fractures. Of non-traumatic disorders, the lower extremities were reported as the injured part in 43% of the disorders. Lumbar spine disorders were the most common non-traumatic disorders, followed by tendon and joint disorders. CONCLUSIONS: Over one-third of orthopedic cases among members of the Japan Self-Defense Forces are traumatic injuries, with the knee being the body part most commonly injured and exercise being the leading cause of injury.

[Matrix metalloproteinases and cartilage matrix degradation in rheumatoid arthritis].

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder characterized by the progressive destruction of articular cartilage. Matrix metalloproteinases (MMPs) constitute a major group among the neutral proteinases that degrade the extracellular matrix of cartilage, including various types of collagen, and aggrecan. Various MMPs are highly produced in synovial fluid and in sera from patients with RA, and are reported to play a pivotal role in cartilage matrix degradation in RA. In this review we describe the members of the MMP family and their basic function, and discuss their role in cartilage destruction in RA.

Comparison of Pedicle Screw Fixation Strength Among Different Transpedicular Trajectories: A Finite Element Study.

STUDY DESIGN: Comparative biomechanical study by finite element (FE) method. OBJECTIVE: To investigate the pullout strength of pedicle screws using different insertional trajectories. SUMMARY OF BACKGROUND DATA: Pedicle screw fixation has become the gold standard for spinal fusion, however, not much has been done to clarify how the fixation strength of pedicle screws are affected by insertional trajectories and bone properties. MATERIALS AND METHODS: Three-dimensional FE models of 20 L4 vertebrae were constructed from the computed tomographic data. Five different transpedicular trajectories were compared: the traditional trajectory, the vertical trajectory, and the 3 lateral trajectories with different sagittal directions (caudal, parallel, cranial). For a valid comparison, screws of the same shape and size were inserted into the same pedicle in each subject, and the pullout strength were compared with nonlinear FE analyses. In addition, the pullout strength was correlated with bone mineral density (BMD). RESULTS: The mean pullout strength showed a 3.9% increase for the vertical trajectory relative to the traditional trajectory, 6.1% for the lateral-caudal trajectory, 21.1% for the lateral-parallel trajectory, and 34.7% for the lateral-cranial trajectory. The lateral-cranial trajectory demonstrated the highest value among all trajectories (P<0.001). In each trajectory, the correlation coefficient between the pullout strength and BMD of the femoral neck (r=0.74-0.83, P<0.01) was higher than the mean BMD of all the lumbar vertebrae (r=0.49-0.75, P<0.01), BMD of the L4 vertebra (r=0.39-0.64, P<0.01), and regional BMD of the L4 pedicle (r=0.53-0.76, P<0.01). CONCLUSIONS: Regional variation in the vertebral bone density and the amount of denser bone-screw interface contribute to the differences of stiffness among different screw trajectories. BMD of the femoral neck is considered to be a better objective predictor of pedicle screw stability than that of the lumbar vertebra.

Upregulated expression of inducible nitric oxide synthase plays a key role in early apoptosis after anterior cruciate ligament injury.

The reason that the anterior cruciate ligament (ACL) has a very poor healing potential after injury is not well understood. In this study, we investigated the role of nitric oxide (NO) in the apoptotic cell death of ACL cells using a rabbit model and in vitro cell culture. The apoptosis of ACL cells in vivo was analyzed by TUNEL assay and electron microscopy. NO synthase (NOS) expression was observed by immunohistochemical analysis. ACL cells were cultured and the susceptibility to NO-induced apoptosis was tested. Inducible NOS (iNOS) expression after treatment with cytokines was examined by immunohistochemical and RT-PCR analyses. Mitogen-activated protein kinase (MAPK) inhibitors were used for the analysis of downstream signals. A significant number of apoptotic cells were observed on days 1 to 3 after injury; the apoptotic rate returned to the control level by day 7. Upregulation of iNOS in the ACL remnant was observed at day 1. Intraarticular injection of NOS inhibitor suppressed the apoptotic rate. Isolated ACL cells showed much higher susceptibility to NO-induced apoptosis than did medial collateral ligament cells. IL-1beta stimulated ACL cells to upregulate iNOS mRNA and increase NO production. p38 MAPK inhibitor decreased NO-induced apoptosis. Rapid iNOS induction after injury contributes to the high apoptotic rate of ACL cells, and this may partly account for the poor healing capacity of this ligament. iNOS and NO production is suggested to be stimulated by IL-1beta, and NO activates the p38 MAPK pathway and triggers an apoptotic signal in ACL cells.

Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived stromal cells using atelocollagen honeycomb-shaped scaffold with a membrane sealing in rabbits.

Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit, have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in plate-culturing and into chondrocytes in an established aggregate culture using defined differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a high density and three-dimensional culture with control medium. An articular cartilage defect was created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation, the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective in repairing articular cartilage defects.

Incidence and Characteristics of Traumatic Shoulder Instability in Japanese Military Cadets.

Little is known regarding the incidence of the shoulder instability in Japan. The aim of this study was to evaluate the incidence of traumatic shoulder instability among Japanese military cadets. A prospective cohort study was performed to capture all traumatic shoulder instability events between 2009 and 2012 among cadets in a military educational academy of the Japan Self Defense Forces. The total number of cadets in the cohort was 5,402 (average age 20.6 years). The incidence of instability events, including dislocation or subluxation, was calculated. Chronicity, demographics of participants, mechanism of injury, and athletic events were also evaluated. The incidence of traumatic dislocation was 4.1/1,000 person-years and that of subluxation was 6.1/1,000 person-years. The incidence of primary dislocation or subluxation was 5.4/1,000 person-years and that of recurrent dislocation or subluxation was 4.7/1,000 person-years. Of first dislocations or subluxations, 92% occurred during sports activities, including after-school sports activities, military training, and gym classes. In conclusion, the overall incidence of shoulder instability events among Japanese military cadets was 10.3/1,000 person-years, and was extremely high. Most shoulder instability events occurred during sports activities, and a program to prevent such injuries during sports activities is necessary for young participants.

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold).

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.

[Mechanism of cartilage destruction in rheumatoid arthritis].

Destruction of cartilage in rheumatoid arthritis (RA) is mediated mainly by proteinases which can degrade cartilage matrix including type II collagen and aggrecan. Of these proteinases, matrix metalloproteinases (MMP) play significant roles in RA pathology, however recent studies show that a disintegrin and metalloproteinase (ADAM) families are another candidates. These proteinases are mainly produced from synovial cells and inflammatory cells, and concentrations of these proteinases in synovial fluid are significantly higher in RA than in OA. Several proteinases have been shown to express in chondorocytes of RA and these chondrocytic proteinases are thought to mediate cartilage destruction in RA. Apoptosis which is mediated by nitric oxide (NO) is another pathway of cartilage destruction in RA.

Therapeutic effect of exendin-4, a long-acting analogue of glucagon-like peptide-1 receptor agonist, on nerve regeneration after the crush nerve injury.

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

Linezolid and vancomycin decrease the therapeutic effect of methylene blue-photodynamic therapy in a mouse model of MRSA bacterial arthritis.

We previously reported that photodynamic therapy (PDT) using intra-articular methylene blue (MB) could be used to treat arthritis in mice caused by bioluminescent methicillin-resistant Staphylococcus aureus (MRSA) either in a therapeutic or in a preventative mode. PDT accumulated neutrophils into the mouse knee via activation of chemoattractants such as inflammatory cytokines or chemokines. In this study, we asked whether PDT combined with antibiotics used for MRSA could provide added benefit in controlling the infection. We compared MB-PDT alone, systemic administration of either linezolid (LZD) alone or vancomycin (VCM) alone or the combination of PDT with either LZD or VCM. Real-time noninvasive imaging was used to serially follow the progress of the infection. PDT alone was the most effective, whereas LZD alone was ineffective and VCM alone showed some benefit. Surprisingly the addition of LZD or VCM reduced the therapeutic effect of PDT alone (P < 0.05). Considering that PDT in this mouse model stimulates neutrophils to be antibacterial rather than actively killing the bacteria, we propose that LZD and VCM might inhibit the activation of inflammatory cytokines without eradicating the bacteria, and thereby reduce the therapeutic effect of PDT.

Photodynamic therapy can induce a protective innate immune response against murine bacterial arthritis via neutrophil accumulation.

BACKGROUND: Local microbial infections induced by multiple-drug-resistant bacteria in the orthopedic field can be intractable, therefore development of new therapeutic modalities is needed. Photodynamic therapy (PDT) is a promising alternative modality to antibiotics for intractable microbial infections, and we recently reported that PDT has the potential to accumulate neutrophils into the infected site which leads to resolution of the infection. PDT for cancer has long been known to be able to stimulate the innate and adaptive arms of the immune system. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, a murine methicillin-resistant Staphylococcus aureus (MRSA) arthritis model using bioluminescent MRSA and polystyrene microparticles was established, and both the therapeutic (Th-PDT) and preventive (Pre-PDT) effects of PDT using methylene blue as photosensitizer were examined. Although Th-PDT could not demonstrate direct bacterial killing, neutrophils were accumulated into the infectious joint space after PDT and MRSA arthritis was reduced. With the preconditioning Pre-PDT regimen, neutrophils were quickly accumulated into the joint immediately after bacterial inoculation and bacterial growth was suppressed and the establishment of infection was inhibited. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of a protective innate immune response against a bacterial pathogen produced by PDT.

Optimal photosensitizers for photodynamic therapy of infections should kill bacteria but spare neutrophils.

Photodynamic therapy (PDT) for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and also by the bactericidal effects of host neutrophils stimulated by PDT. Therefore, PDT-induced damage to neutrophils must be minimized, while direct photoinactivation of bacteria is maintained to maximize the therapeutic efficacy of antimicrobial PDT in vivo. However, there has been no study in which the cytocidal effect of PDT on neutrophils was investigated. In this study, the cytocidal effects of PDT on neutrophils were evaluated using different antimicrobial photosensitizers to find suitable candidate photosensitizers for antimicrobial PDT. PDT on murine peripheral-blood neutrophils was performed in vitro using each photosensitizer at a concentration that exerted a maximum bactericidal effect on methicillin-resistant Staphylococcus aureus, and morphological alteration and viability of neutrophils were studied. Most neutrophils were viable (>80%) after PDT using toluidine blue-O (TB) or methylene blue (MB), while neutrophils showed morphological change and their viabilities were decreased (<70%) after PDT using other photosensitizers (erythrosine B, rose bengal, crystal violet, Photofrin, new methylene blue and Laserphyrin). These results suggest that PDT using TB or MB can preserve host neutrophils while exerting a significant therapeutic effect on in vivo localized microbial infection.

Cortical layer V neurons in the auditory and visual cortices of normal, reeler, and yotari mice.

Both in the Reelin-deficient reeler and Dab1-deficient yotari mice, layer V corticospinal tract neurons in the sensory-motor cortex are radially spread instead of being confined to a single cortical layer. In the present study, we examined distribution pattern of cortical layer V neurons in the visual and auditory cortices of reeler and yotari mice with the injection of HRP into the superior and inferior colliculi of the adult animals, respectively. After the injection of HRP into the superior colliculus of the normal mouse, retrogradely labeled cells were distributed in layer V of the visual cortex, while the similar injection of HRP in the reeler and yotari mice produced radial dispersion of retrograde labeling through all of the depths of the visual cortex of these mutant mice. Next, we injected HRP into the inferior colliculus of the normal, reeler and yotari mice. Retrogradely labeled neurons were distributed in layer V of the normal auditory cortex, whereas they were again radially scattered in the auditory cortex of the reeler and yotari mice. Taken together with the previous and present findings, layer V cortical efferent neurons are radially scattered in the sensory-motor, visual and auditory cortices of the reeler and yotari mice.

Influence of intra-articular neutrophils on the effects of photodynamic therapy for murine MRSA arthritis.

Although there have been some reports about the cytotoxic effects of photodynamic therapy (PDT) on multidrug-resistant bacteria, there have been few reports in which favorable results of PDT on a local infection site are described. This study aimed to verify the hypothesis that the low efficacy of PDT on a local infection site is due to the cytotoxic effect of PDT on leukocytes. PDT using Photofrin exerted significant cytotoxicity for cultured methicillin-resistant Staphylococcus aureus (MRSA). Nevertheless, this therapeutic modality was not effective for a murine MRSA arthritis model. Approximately 30% of intra-articular leukocytes, mainly neutrophils, died immediately after PDT, and a further decrease in the number of intra-articular leukocytes and atrophy of the synovial tissue were seen 24 h after PDT. Isolated peripheral neutrophils showed significant affinity for Photofrin and showed significant morphological damage, resulting in cell death, when they were subject to PDT using Photofrin. These results indicate that intra-articular neutrophils have an influence on the effects of PDT for MRSA arthritis.