Enhanced immunity in intradermal vaccination by novel hollow microneedles.

BACKGROUND: The intradermal (ID) route for vaccination represents an effective alternative to subcutaneous (SC)/intramuscular administration to induce protective immunity. However, a critical issue associated with ID vaccination is the precise delivery of solution in the upper dermis, which ensures enhanced immunity. METHODS: We fabricated a hollow microneedle unit made of poly-glycolic acid by injection molding and bonding, and created a dedicated prototype injector. To ensure ID delivery of solution, the injected site was macroscopically and microscopically examined. Serum immunoglobulin G antibody production was measured by enzyme immunoassay and compared in groups of rats following either ID delivery with microneedles or SC administration with a 27-G stainless needle of graded vaccine doses. RESULTS: The unit used a tandem array of six microneedles, each with a side delivery hole, and a conduit inside for solution. Microneedles installed in the injector punctured the skin with the aid of a spring. Injection of solution formed a wheal due to ID distribution. Histologically, a wedge-shaped skin defect in the upper skin corresponded to each puncture site. Antibody titers following vaccinations on days 1 and 8 were significantly higher with ID injection than with SC delivery on day 15 and every 7 days thereafter until day 36 with mumps vaccination, and until day 36 with varicella vaccination. CONCLUSIONS: The microneedle unit presented here delivered solution intradermally without any difficulty and evoked antibody responses against viruses even with the reduced vaccine volume. Our findings confirm promising results of ID delivery as an immunogenic option to enhance vaccination efficacy.

A long-term follow-up study of open reduction using Ludloff's approach for congenital or developmental dislocation of the hip.

OBJECTIVES: There are several reports clarifying successful results following open reduction using Ludloff's medial approach for congenital (CDH) or developmental dislocation of the hip (DDH). This study aimed to reveal the long-term post-operative course until the period of hip-joint maturity after the conventional surgical treatments. METHODS: A long-term follow-up beyond the age of hip-joint maturity was performed for 115 hips in 103 patients who underwent open reduction using Ludloff's medial approach in our hospital. The mean age at surgery was 8.5 months (2 to 26) and the mean follow-up was 20.3 years (15 to 28). The radiological condition at full growth of the hip joint was evaluated by Severin's classification. RESULTS: All 115 hips successfully attained reduction after surgery; however, 74 hips (64.3%) required corrective surgery at a mean age of 2.6 years (one to six). According to Severin's classification, 69 hips (60.0%) were classified as group I or II, which were considered to represent acceptable results. A total of 39 hips (33.9%) were group III and the remaining seven hips (6.1%) group IV. As to re-operation, 20 of 21 patients who underwent surgical reduction after 12 months of age required additional corrective surgeries during the growth period as the hip joint tended to subluxate gradually. CONCLUSION: Open reduction using Ludloff's medial approach accomplished successful joint reduction for persistent CDH or DDH, but this surgical treatment was only appropriate before the ambulating stage. Cite this article: Bone Joint Res 2014;3:1-6.

Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger.

Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H(2)O(2)) to H(2)O and O(2). In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H(2)O(2) and O(2) without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H(2)O(2) using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H(2)O(2) are significant events for monocyte/macrophage differentiation by TPA.

Follow-up study of conservative treatment for atlantoaxial rotatory displacement.

Rotatory displacement of the atlantoaxial joint is one of the causes of torticollis in children. Some of these cases show persistent symptoms and may lead to rotatory fixation; however, only a few studies have been directed to the prognosis of atlantoaxial rotatory displacement after conservative treatment. Clinical and radiographic reviews were performed in 35 patients (16 male and 19 female) with rotatory displacement of the atlantoaxial joint. The mean age at admission was 6.5 (range: 2-12) years old, and the mean follow-up period was 3.4 (1.4-5.8) years. All patients successfully achieved reduction after 2 to 3 weeks of continuous halter traction; however, 9 patients (25.7%) experienced recurrence, and 7 patients (20.0%) had a slight torticollis at follow-up. The duration of symptoms before treatment affected the recurrence rate, and the torticollis was apt to remain in cases with severe displacement at admission.

Cervical posterior fusion with wave-shaped rod under local anesthesia for cervical spondylotic myelopathy: review of 12 patients.

STUDY DESIGN: Clinical evaluation of cervical interspinous fusion under local anesthesia in elderly patients with cervical spondylotic myelopathy. OBJECTIVES: To evaluate the effectiveness of cervical posterior fusion with wave-shaped rods inserted under local anesthesia for elderly high-risk patients with cervical spondylotic myelopathy. SUMMARY OF BACKGROUND DATA: A substantial number of patients cannot undergo surgical interventions under general anesthesia because of their general medical complications. Although such patients would become unable to walk, which might induce a worsening of their general condition, conservative treatments had been adopted as the only treatment for these patients. The authors have obtained satisfactory results by means of posterior interspinous fusion under local anesthesia even in the high-risk patients with severe cervical spondylotic myelopathy. The aims of this surgical technique were to adjust cervical alignment and to stabilize the motion segment(s) without decompression. PATIENTS AND METHODS: Between May 1989 and August 1998, 12 elderly patients (3 men and 9 women) with cervical spondylotic myelopathy were treated with posterior interspinous fusion using wave-shaped rods inserted under local anesthesia. The average age at the surgery was 76.9 years. The average follow-up period was 5 years 6 months. All patients were unable to walk without any assistance because of their advanced myelopathy. It was felt that all of them would be unable to accept general anesthesia because of their generally poor medical conditions. Preoperative severity of the clinical symptoms and postoperative recovery were evaluated by a scoring system proposed by the Japanese Orthopaedic Association, which had 17 points at full mark. RESULTS: The average duration of the surgical procedure was 122.8 minutes. The average total blood loss was 118.6 g. No instrument failures were denoted. Neither neural deterioration nor major complication was observed relating to the surgery. Radiographic bony union of the grafted bone was achieved in all patients. Progression of myelopathy was arrested in all 12 patients, and clinical symptoms were improved in 10 patients. The mean Japanese Orthopaedic Association scores had increased from 5.0 to 10.2 points. CONCLUSIONS: Twelve high-risk patients with cervical spondylotic myelopathy were treated with posterior interspinous fusion using wave-shaped rods inserted under local anesthesia. This method was evaluated as an effective surgical salvage without any mortal complications even in the elderly high-risk patients.

Two hyperandrogenic adolescent girls with congenital portosystemic shunt.

UNLABELLED: We describe two adolescent girls with a congenital portosystemic shunt who exhibited hyperandrogenism in addition to insulin resistant hyperinsulinaemia. Case 1 was referred to our clinic to undergo a routine clinical work-up prior to tonsillectomy at 14 years of age. Mild liver dysfunction was identified and hypogenesis of the portal vein with a congenital portosystemic shunt diagnosed. Primary amenorrhoea and virilization were evident and an endocrinological evaluation revealed hyperandrogenism and insulin resistant hyperinsulinaemia. Case 2 was referred at 15 years of age because of cardiomegaly. Mild liver dysfunction and hyperbilirubinaemia led to a diagnosis of agenesis of the portal vein with a congenital portosystemic shunt. Virilization was evident and an endocrinological evaluation revealed hyperandrogenism and insulin resistant hyperinsulinaemia. The haemodynamics of these patients were similar to those of secondary portosystemic shunt due to liver cirrhosis, which is often associated with hyperinsulinaemia and/or non-insulin dependent diabetes mellitus. On the other hand, hyperandrogenism is associated with certain insulin-resistant conditions with hyperinsulinaemia, including the polycystic ovary syndrome (PCO). Hyperinsulinaemia is believed to cause hyperandrogenism in patients with PCO by stimulating androgen production in both the ovary and adrenal gland. Therefore, in congenital portosystemic shunts, hyperinsulinaemia is also thought to cause hyperandrogenism due to the same mechanism. CONCLUSION: A certain percentage of female patients with hyperandrogenism, likely including those with polycystic ovary syndrome may also have congenital portosystemic shunts. Our results indicate that serum levels of total bile acids and ammonia are prognostic indicators of this hepatic vascular anomaly.

TNF-alpha induced p21(WAF1) but not Bax in colon cancer cells WiDr with mutated p53: important role of protein stabilization.

The p21(WAF1)induces cell cycle arrest at G(1)and its expression is regulated by the functional p53. TNF-alpha induced expression of p21(WAF1)at protein and mRNA levels in a dose-dependent fashion with an association with G(1)-arrest in human colon cancer cells WiDr that carry mutated p53 at codon 273 (His(273)). However, TNF-alpha did not affect the levels of the Bax protein, which also has p53-binding sites on its promoter and causes apoptosis. Further experiments suggested that cycloheximide (CHX), a protein synthesis inhibitor, increased the levels of p21(WAF1)mRNA and the induction of p21(WAF1)mRNA by TNF-alpha did not require new protein synthesis. Co-transfection of the p53 His(273)expression construct with a luciferase gene controlled by the p21(WAF1)promoter showed that the p53 His(273)was inactive, although TNF-alpha increased the transcriptional rate of p21(WAF1)in these cells. Further study found that TNF-alpha markedly stabilized the p21(WAF1)protein. These findings suggest that TNF-alpha induces expression of p21(WAF1)through a distinct pathway from Bax and that protein stabilization is an important mechanism in the expression of p21(WAF1)independent of p53.

IL-1 induces expression of p21(WAF1) independently of p53 in high-passage human embryonic fibroblasts WI38.

We tested the effect of IL-1 on the expression of p21(WAF1) in human embryonic fibroblasts WI38. Exposure to IL-1 caused induction of p21(WAF1) protein in high-passage WI38 cells but not in early-passage cells. However, IL-1 did not stimulate the transcription of a CAT-reporter gene having two copies of the p53-responsive element on its promoter or the p53-binding capacity of nuclear extracts, although it increased transcriptional rate of p21(WAF1) in these high-passage cells. These results suggest that the induction of p21(WAF1) by IL-1 occurs at the transcriptional level, but p53 function is not required in these cells. Further studies found that IL-1 did not cause cell-cycle arrest, and the overexpression of p21(WAF1) resulted in only a slight delay of cell growth, while the level of p21(WAF1) coprecipitated with cyclin-dependent kinase-2 (Cdk2) was increased by IL-1. Moreover, a kinase assay of Cdk2 immunoprecipitates showed that IL-1 did not reduce the kinase activity, and IL-1 did not affect the status of phosphorylation of the retinoblastoma gene product (Rb). These findings imply that despite the induction of p21(WAF1), this cannot fully account for the growth arrest in high-passage WI38 cells. Thus, IL-1 mediates p21(WAF1) induction through a p53-independent pathway(s) in high-passage WI38 cells, but the cell cycle is regulated independently of p21(WAF1).

Cervical myelopathy caused by C3-C4 spondylosis in elderly patients: a radiographic analysis of pathogenesis.

STUDY DESIGN: A radiographic analysis of elderly patients with cervical spondylotic myelopathy, particularly those with involvement of the C3-C4 level. OBJECTIVES: To elucidate the potential contributors to the higher incidence of pathology at C3-C4 in the elderly. SUMMARY OF BACKGROUND DATA: In this study, the elder patients showed a greater predilection for involvement of the C3-C4 lesion compared with their younger counterparts. No previous study has addressed C3-C4 pathology in elderly patients. METHODS: This study included 18 patients, 10 men and 8 women, with cervical spondylotic myelopathy caused by C3-C4 disorders (group I). For the purpose of comparison, 18 younger patients (less than 50 years of age) with myelopathy (group II) and 30 volunteers over the age of 65 (group III) were also investigated. Mean age at admission was 73.5 years for group I, 42.4 years for group II, and 73.4 years for group III. Radiographic analysis, using static and dynamic radiographs, was performed to evaluate the morphologic features. RESULTS: The mean spinal canal diameter for groups I and II was significantly smaller than that for group III. Group I exhibited greater C2-C7 lordosis. The aged population, group I and group III, showed greater C3-C4 angulation associated with C4 forward inclination in neutral standing position as compared with younger patients. Regarding dynamic factors, group I showed the largest segmental motion at C3-C4, and, conversely, the smallest mobility at the lower segments, with significant differences. CONCLUSIONS: Using radiographic analysis, morphologic features that predispose patients to disorders of the C3-C4 motion segment were evaluated. These features included 1) greater C3-C4 angulation associated with age-related postural change and 2) hypermobility at the C3-C4 segment compensating for decreased mobility at the lower segments.

Role of TNFalpha in regulation of myeloperoxidase expression in irradiated HL60 promyelocytic cells.

Irradiation increases the generation of reactive oxygen intermediates, including hydrogen peroxide (H(2)O(2)). Myeloperoxidase (MPO), a heme-containing glycoprotein located in the primary granules of polymorphonuclear leukocytes and monocytes, reacts with H(2)O(2) and halide ion and produces a more potent microbicidal oxidant, hypochlorous acid (HOCl). Human HL60 promyelocytes constitutively had high levels of MPO protein and mRNA. Irradiation decreased the levels of MPO transcripts; the decrease in MPO transcripts by irradiation occurred in an almost dose-dependent manner. HL60 cells produce tumor necrosis factor alpha (TNFalpha), and irradiation markedly increased the TNFalpha production in these cells; in turn, TNFalpha decreased the levels of MPO transcripts in these cells. Furthermore, treatment of cells with anti-TNFalpha antibody blocked the reduction of MPO by irradiation. We also found that irradiation decreased the levels of the MPO mRNA with concomitant increased levels of TNFalpha mRNA in differentiation-induced HL60 cells and human THP-1 monocytic cells. Irradiation reduced the rate of MPO transcription but had only a slight effect on the half-life of MPO mRNA in HL60 cells. Our results suggest that irradiation reduces the steady-state levels of MPO mRNA mainly at transcriptional level and the endogenous production of TNFalpha is required for the reduction by irradiation in HL60 cells.

Genetic variation in the capsid region of human astrovirus serotype 4 isolated in Japan.

The entire capsid regions of 12 serotype-4 astroviruses from Japan were sequenced and compared with those of other serotypes. Serotype-4 isolates were divided into two new subgroups. The intrasubgroup nucleic acid and deduced amino acid sequences were quite homologous (more than 93%), but slightly less so between subgroups (almost 85%). However, the serotype-4 sequences differed from those of serotypes 1, 2, 3, 5, 6, 7 and 8 (less than 50%). Determining whether these differences significantly alter the epidemiology and antigenicity will require further investigation.

Chromosome aberrations in bone marrow cells from Japanese patients with thorotrastosis.

Exposure of bone marrow cells to alpha-particle radiation causes various types of chromosome abnormalities and hematological malignancies. We performed chromosome analysis of hematopoietic stem cells from the bone marrow of 52 Japanese patients with thorotrastosis and 21 age-matched controls. The frequency of cells with stable chromosome abnormalities was significantly higher in the patients with thorotrastosis. Further studies found 14 clonal chromosome aberrations in cells from 11 patients (21.2%); clones observed in the cells from 2 of these patients had high frequencies of chromosome abnormalities. In one case, 68 to 100% of the cells analyzed had a large partial loss in the short arm of chromosome 1 and a translocation between the short arms of chromosomes 2 and 3 [46,XY,1p-,t(2p+;3p-)]. The cells from the other patient contained a clone with partial loss of both the short and long arms of chromosome 5 (46,XX,5p-,5q-). The frequency of this clone has been constant for the last 15 years (6-24%). We also analyzed bone marrow mononuclear cells from 17 of the patients for mutations of the TP53 tumor suppressor gene (formerly known as p53). However, no mutation was found in any of the cells, including those from the 2 patients with abnormal clones. Moreover, repeated medical examinations showed no evidence of leukemia or myelodysplasia in these patients. Our study suggests that exposure of bone marrow cells to alpha-particle radiation may induce clonal chromosomal aberrations at a high frequency.

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis is mediated by tumor necrosis factor alpha in human monocytic U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-alpha (TNFalpha) in U937 cells, and exogenously added TNFalpha induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFalpha antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFalpha and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFalpha in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFalpha is an autocrine-stimulating factor for the induction of apoptosis in these cells.

p21WAF1 expression by an activator of protein kinase C is regulated mainly at the post-transcriptional level in cells lacking p53: important role of RNA stabilization.

p21(WAF1) inhibits cyclin-cyclin-dependent kinase (Cdk) complexes, causing cell cycle arrest. p21(WAF1) contains p53-binding sites in its promoter and expression of p21(WAF1) is induced by functional p53. In the present work, we have studied the role of protein kinase C (PKC) in the induction of p21(WAF1) and show that induction of p21(WAF1) expression can occur by activation of PKC in cells having no p53. Human ovarian carcinoma cells, SKOV-3, lack p53 protein and PMA, a potent activator of PKC, did not induce p53. PMA increased the expression of p21(WAF1) mRNA both in these cells and in other cells which do not contain p53 (THP-1 and U937). Treatment of human embryonic fibroblasts, WI38, with PMA also induced the accumulation of p21(WAF1) without affecting p53 levels. However, PMA did not increase levels of p21(WAF1) mRNA in cells where either the PKC or the mitogen-activated protein kinase pathway was blocked. Furthermore, treatment of cells with various phorbol ester derivatives which activate PKC resulted in the induction of p21(WAF1) in SKOV-3 cells. In contrast, phorbol esters which do not activate PKC failed to induce p21(WAF1) expression. PMA increased the transcriptional rate of p21(WAF1) and activated the transcription of a luciferase reporter gene, controlled by the p21 promoter, in SKOV-3 cells with or without a p53 consensus-binding sequence. By contrast, PMA markedly stabilized p21(WAF1) mRNA; the half-life (t1/2) of p21(WAF1) in PMA-treated cells was >8 h compared with <1 h in untreated cells. These findings provide evidence that the PKC pathway induces expression of p21(WAF1) independently of p53. Our present study also suggests that the accumulation of p21(WAF1) transcripts by PMA occurs mainly at post-transcriptional level.

Determination of serotypes of astroviruses by reverse transcription-polymerase chain reaction and homologies of the types by the sequencing of Japanese isolates.

Human standard astroviruses, serotypes 1 to 7, and 35 Japanese isolates were typed by reverse transcription and polymerase chain reaction (RT-PCR) with serotype-specific primers for the first time. The results were identical with those obtained by enzyme immunoassay with serotype-specific polyclonal antibodies, a method which has already been reported. RT-PCR with serotype-specific primers is useful for epidemiological studies of astroviruses where serotype-specific polyclonal antibodies are not available. Two parts of the capsid region, N terminus and C terminus, were sequenced. Serotypes differed in those regions. The N terminus differed less than the C terminus between serotypes. Both the N terminus and C terminus were similar intraserotypically with the exception of serotype-4 isolates which could be divided into A and B subgroups on the basis of their C terminus sequences, which were not known previously.

Relationship between LET and RBE values for Escherichia coli determined using carbon ion beams from the TIARA cyclotron and HIMAC synchrotron.

NASA: Researchers studied the effects of ion beams on cell lethality in two strains of Escherichia coli. Experiments were conducted on the wild-type strain and a DNA repair-deficient mutant strain that lacks the ability to repair DNA damage. A final aspect of the study was to examine the relationship between the linear energy transfer and relative biological effectiveness values for E. coli cell lethality and dose-response for decreasing the survival fraction to 10 percent.^ieng

Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges superoxide (O2-) ions. We studied the regulation of MnSOD gene expression by irradiation and the mechanisms in human monocytic cell line THP-1. We found that irradiation induced expression of the MnSOD gene through the autocrine mechanism, involving the production of tumour necrosis factor (TNF). Irradiation increased TNF production in THP-1 cells, and TNF increased the levels of MnSOD transcripts. Supernatant from irradiated THP-1 cells induced the expression of MnSOD mRNA, and anti-TNF antibody blocked the induction of MnSOD mRNA. Irradiation also increased the levels of MnSOD mRNA in other myelocytic cell lines, HL60 and KG-1, and the ovarian cancer cell line SK-OV-3. Moreover, increased levels of MnSOD mRNA were observed in mature myeloid cells, including macrophages and granulocytes, as well as in immature cells. However, irradiation did not increase the level of MnSOD mRNA in THP-1 cells with prolonged exposure to PMA. We also found that irradiation increased the rate of MnSOD transcription, and irradiation stabilized MnSOD mRNA in THP-1 cells. Our results indicate that the endogenous production of TNF is required, at least in part, for the induction of MnSOD mRNA expression by irradiation in THP-1 cells, and the increased levels of MnSOD transcripts on irradiation occur through a pathway involving protein kinase C activation. Our results also indicate that the increase in MnSOD mRNA caused by irradiation is regulated by both transcriptional and post-transcriptional mechanisms.

Anti-cancer agent OK432 induces manganese superoxide dismutase in human granulocytes.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in scavenging O2-. OK432, a Streptococcal preparation, has anti-tumor activity and is also known to be a biological-response modifier. In this study, we have examined the regulation of MnSOD gene by OK432 in human granulocytes. Granulocytes had a low activity of MnSOD; OK432 increased MnSOD mRNA levels in a dose-dependent manner and its activity in granulocytes. OK432 also induced the MnSOD mRNA in HL60 promyelocytes, while not affecting the levels of MnSOD mRNA in human fibroblasts. Pre-treatment with either actinomycin D or cycloheximide inhibited the induction of MnSOD mRNA by OK432. OK432 increased the levels of interleukin-1 (IL-1) and tumor-necrosis-factor-alpha (TNFalpha) mRNA in granulocytes. Furthermore, the induction of MnSOD mRNA by OK432 was blocked by anti-IL-1 antibody but not by anti-TNF antibody. These results suggest that the induction of MnSOD mRNA by OK432 is regulated at the transcriptional level, and OK432 induces MnSOD mRNA, at least in part, through production of IL-1 in granulocytes.