Effect of sustained PDGF nonviral gene delivery on repair of tooth-supporting bone defects.

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) promotes soft tissue and bone healing, and is Food and Drug Administration-approved for treatment of diabetic ulcers and periodontal defects. The short half-life of topical rhPDGF-BB protein application necessitates bolus, high-dose delivery. Gene therapy enables sustained local growth factor production. A novel gene activated matrix delivering polyplexes of polyethylenimine (PEI)-plasmid DNA encoding PDGF was evaluated for promotion of periodontal wound repair in vivo. PEI-pPDGF-B polyplexes were tested in human periodontal ligament fibroblasts and human gingival fibroblasts for cell viability and transfection efficiency. Collagen scaffolds containing PEI-pPDGF-B polyplexes at two doses, rhPDGF-BB, PEI vector or collagen alone were randomly delivered to experimentally induced tooth-supporting periodontal defects in a rodent model. Mandibulae were collected at 21 days for histologic observation and histomorphometry. PEI-pPDGF-B polyplexes were biocompatible to cells tested and enzyme-linked immunosorbent assay confirmed the functionality of transfection. Significantly greater osteogenesis was observed for collagen alone and rhPDGF-BB versus the PEI-containing groups. Defects treated with sustained PDGF gene delivery demonstrated delayed healing coupled with sustained inflammatory cell infiltrates lateral to the osseous defects. Continuous PDGF-BB production by nonviral gene therapy could have delayed bone healing. This nonviral gene delivery system in this model appeared to prolong inflammatory response, slowing alveolar bone regeneration in vivo.

3D osteoarthritic changes in TMJ condylar morphology correlates with specific systemic and local biomarkers of disease.

OBJECTIVE: To assess 3D morphological variations and local and systemic biomarker profiles in subjects with a diagnosis of temporomandibular joint osteoarthritis (TMJ OA). DESIGN: Twenty-eight patients with long-term TMJ OA (39.9 ± 16 years), 12 patients at initial diagnosis of OA (47.4 ± 16.1 years), and 12 healthy controls (41.8 ± 12.2 years) were recruited. All patients were female and had cone beam CT scans taken. TMJ arthrocentesis and venipuncture were performed on 12 OA and 12 age-matched healthy controls. Serum and synovial fluid levels of 50 biomarkers of arthritic inflammation were quantified by protein microarrays. Shape Analysis MANCOVA tested statistical correlations between biomarker levels and variations in condylar morphology. RESULTS: Compared with healthy controls, the OA average condyle was significantly smaller in all dimensions except its anterior surface, with areas indicative of bone resorption along the articular surface, particularly in the lateral pole. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were significantly correlated with bone apposition of the condylar anterior surface. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFßb1, IFNγg, TNFαa, IL-1αa, and IL-6 were significantly correlated with flattening of the lateral pole. Expression levels of ANG were significantly correlated with the articular morphology in healthy controls. CONCLUSIONS: Bone resorption at the articular surface, particularly at the lateral pole was statistically significant at initial diagnosis of TMJ OA. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 were correlated with bone apposition. Serum levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFß1, IFNγ, TNFα, IL-1α, and IL-6 were correlated with bone resorption.

LIM domain protein-3 (LMP3) cooperates with BMP7 to promote tissue regeneration by ligament progenitor cells.

Gene transfer of key regulators of osteogenesis for mesenchymal stem cells represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. As little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP (Alkaline Phosphatase), BSP (Bone Sialoprotein) and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides the evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.

Inhibition of Mertk Signaling Enhances Bone Healing after Tooth Extraction.

Regeneration of alveolar bone is an essential step in restoring healthy function following tooth extraction. Growth of new bone in the healing extraction socket can be variable and often unpredictable when systemic comorbidities are present, leading to the need for additional therapeutic targets to accelerate the regenerative process. One such target is the TAM family (Tyro3, Axl, Mertk) of receptor tyrosine kinases. These proteins have been shown to help resolve inflammation and maintain bone homeostasis and thus may have therapeutic benefits in bone regeneration following extraction. Treatment of mice with a pan-TAM inhibitor (RXDX-106) led to accelerated alveolar bone fill following first molar extraction in a mouse model without changing immune infiltrate. Treatment of human alveolar bone mesenchymal stem cells with RXDX-106 upregulated Wnt signaling and primed the cells for osteogenic differentiation. Differentiation of human alveolar bone mesenchymal stem cells with osteogenic media and TAM-targeted inhibitor RXDX-106 (pan-TAM), ASP-2215 (Axl specific), or MRX-2843 (Mertk specific) showed enhanced mineralization with pan-TAM or Mertk-specific inhibitors and no change with Axl-specific inhibitor. First molar extractions in Mertk-/- mice had increased alveolar bone regeneration in the extraction socket relative to wild type controls 7 d postextraction. Flow cytometry of 7-d extraction sockets showed no difference in immune cell numbers between Mertk-/- and wild type mice. RNAseq of day 7 extraction sockets showed increased innate immune-related pathways and genes associated with bone differentiation in Mertk-/- mice. Together, these results indicate that TAM receptor signaling, specifically through Mertk, can be targeted to enhance bone regeneration after injury.

Multicompartmental Scaffolds for Coordinated Periodontal Tissue Engineering.

Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue-to-bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

Maresin 1 Promotes Wound Healing and Socket Bone Regeneration for Alveolar Ridge Preservation.

Tooth extraction results in alveolar bone resorption and is accompanied by postoperative swelling and pain. Maresin 1 (MaR1) is a proresolving lipid mediator produced by macrophages during the resolution phase of inflammation, bridging healing and tissue regeneration. The aim of this study was to examine the effects of MaR1 on tooth extraction socket wound healing in a preclinical rat model. The maxillary right first molars of Sprague-Dawley rats were extracted, and gelatin scaffolds were placed into the sockets with or without MaR1. Topical application was also given twice a week until complete socket wound closure up to 14 d. Immediate postoperative pain was assessed by 3 scores. Histology and microcomputed tomography were used to assess socket bone fill and alveolar ridge dimensional changes at selected dates. The assessments of coded specimens were performed by masked, calibrated examiners. Local application of MaR1 potently accelerated extraction socket healing. Macroscopic and histologic analysis revealed a reduced soft tissue wound opening and more rapid re-epithelialization with MaR1 delivery versus vehicle on socket healing. Under micro-computed tomography analysis, MaR1 (especially at 0.05 µg/µL) stimulated greater socket bone fill at day 10 as compared with the vehicle-treated animals, resulting in less buccal plate resorption and a wider alveolar ridge by day 21. Interestingly, an increased ratio of CD206+:CD68+ macrophages was identified in the sockets with MaR1 application under immunohistochemistry and immunofluorescence analysis. As compared with the vehicle therapy, local delivery of MaR1 reduced immediate postoperative surrogate pain score panels. In summary, MaR1 accelerated extraction wound healing, promoted socket bone fill, preserved alveolar ridge bone, and reduced postoperative pain in vivo with a rodent preclinical model. Local administration of MaR1 offers clinical potential to accelerate extraction socket wound healing for more predictable dental implant reconstruction.

Minimally Invasive Approach for Diagnosing TMJ Osteoarthritis.

This study's objectives were to test correlations among groups of biomarkers that are associated with condylar morphology and to apply artificial intelligence to test shape analysis features in a neural network (NN) to stage condylar morphology in temporomandibular joint osteoarthritis (TMJOA). Seventeen TMJOA patients (39.9 ± 11.7 y) experiencing signs and symptoms of the disease for less than 10 y and 17 age- and sex-matched control subjects (39.4 ± 15.2 y) completed a questionnaire, had a temporomandibular joint clinical exam, had blood and saliva samples drawn, and had high-resolution cone beam computed tomography scans taken. Serum and salivary levels of 17 inflammatory biomarkers were quantified using protein microarrays. A NN was trained with 259 other condyles to detect and classify the stage of TMJOA and then compared to repeated clinical experts' classifications. Levels of the salivary biomarkers MMP-3, VE-cadherin, 6Ckine, and PAI-1 were correlated to each other in TMJOA patients and were significantly correlated with condylar morphological variability on the posterior surface of the condyle. In serum, VE-cadherin and VEGF were correlated with one another and with significant morphological variability on the anterior surface of the condyle, while MMP-3 and CXCL16 presented statistically significant associations with variability on the anterior surface, lateral pole, and superior-posterior surface of the condyle. The range of mouth opening variables were the clinical markers with the most significant associations with morphological variability at the medial and lateral condylar poles. The repeated clinician consensus classification had 97.8% agreement on degree of degeneration within 1 group difference. Predictive analytics of the NN's staging of TMJOA compared to the repeated clinicians' consensus revealed 73.5% and 91.2% accuracy. This study demonstrated significant correlations among variations in protein expression levels, clinical symptoms, and condylar surface morphology. The results suggest that 3-dimensional variability in TMJOA condylar morphology can be comprehensively phenotyped by the NN.

Platelet-derived growth factor-B gene delivery sustains gingival fibroblast signal transduction.

BACKGROUND AND OBJECTIVE: Platelet-derived growth factor-BB is a potent mediator of tooth-supporting periodontal tissue repair and regeneration. A limitation of the effects of topical platelet-derived growth factor-BB application is its short half-life in vivo. Gene therapy has shown strong promise for the long-term delivery of platelet-derived growth factor in both skin ulcer healing and periodontal tissue engineering. However, little is known regarding the extended effects of platelet-derived growth factor-B on cell signaling via gene delivery, especially at the level of phosphorylation of intracellular kinases. This study sought to evaluate the effect of gene transfer by Ad-PDGF-B on human gingival fibroblasts (HGFs) and the subsequent regulation of genes and cell-surface proteins associated with cellular signaling. MATERIAL AND METHODS: HGFs from human subjects were treated by adenoviral PDGF-B, PDGF-1308 (a dominant negative mutant of PDGF) and recombinant human platelet-derived growth factor-BB, and then incubated in serum-free conditions for various time points and harvested at 1, 6, 12, 24, 48, 72 and 96 h. Exogenous PDGF-B was measured by RT-PCR and Western blot. Cell proliferation was evaluated by [methyl-3H]thymidine incorporation assay. We used proteomic arrays to explore phosphorylation patterns of 23 different intracellular kinases after PDGF-B gene transfer. The expression of alpha and beta PDGFR and Akt were measured by Western blot analysis. RESULTS: Sustained in vitro expression of PDGF-B in HGFs by Ad-PDGF-B transduction was seen at both the mRNA and protein levels. Compared to rhPDGF-BB and Ad-PDGF-1308, Ad-PDGF-B maintained cell growth in serum-free conditions, with robust increases in DNA synthesis. Gene delivery of PDGF-B also prolonged downregulation of the growth arrest specific gene (gas) PDGF alpha R. Of the 23 intracellular kinases that we tested in proteomic arrays, Akt revealed the most notable long-term cell signaling effect as a result of the over-expression of Ad-PDGF-B, compared with pulse recombinant human platelet-derived growth factor BB. Prolonged Akt phosphorylation was induced by treatment with Ad-PDGF-B, for at least up to 96 h. CONCLUSION: These findings further demonstrate that gene delivery of PDGF-B displays sustained signal transduction effects in human gingival fibroblasts that are higher than those conveyed by treatment with recombinant human platelet-derived growth factor-BB protein. These data on platelet-derived growth factor gene delivery contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.

Characterization of vascular leak syndrome induced by the toxin component of Pseudomonas exotoxin-based immunotoxins and its potential inhibition with nonsteroidal anti-inflammatory drugs.

Clinical trials of immunotoxins in cancer patients have been limited in many cases by vascular leak syndrome (VLS). Recently, rats were identified as a model for VLS induced by BR96 sFv-PE40, a carcinoma-reactive single-chain immunotoxin. In this study, the toxin component of this immunotoxin, PE40, was found to be responsible for inducing hydrothorax in rats, thereby demonstrating that direct binding to the BR96 antigen was not essential to the onset of VLS. Mutational analysis of PE40 determined that both ADP ribosylation and proteolytic processing functions innate to Pseudomonas exotoxin A (PE) were necessary for PE40 to induce hydrothorax in rats; however, neither function by itself was sufficient for VLS induction. Additionally, nonsteroidal anti-inflammatory agents were found to block VLS in rats receiving BR96 sFv-PE40. These results demonstrate that the toxin component of PE-based immunotoxins induce VLS and suggest agents for clinical management of the toxicity.

Range of ventricular ectopic complexes in healthy subjects studied with repeated ambulatory electrocardiographic recordings.

The incidence and forms of ventricular premature complexes (VPCs) in apparently healthy subjects were studied to determine long-term reproducibility of day to day variation on Holter electrocardiogram. The study included 152 men and 68 women ranging in age from 20 to 78 years who were having routine check-ups that revealed no cardiovascular abnormalities. In addition to routine measurements, Holter electrocardiography was recorded during daily life, and the total number of VPCs occurring during 24 hours was visually calculated on replayed electrocardiographic tracings. Forms of VPCs and incidence of VPCs in 10-year age groups of the subjects were also recorded. No VPCs were observed in 56% of the subjects; 93% showed less than 50 VPCs and the other 7% of the 220 subjects had greater than or equal to 50 VPCs. Forty-one of the 220 subjects returned for routine follow-up 1 year later. Repeated Holter electrocardiograms at this time showed high reproducibility of less than 50 VPCs. A small number of multiform VPCs were reproducible but paired VPCs were not. Physiologic definition of VPCs in healthy subjects appears to be clinically significant.

New method for determining prognosis of patients with psoriasis (E-PAP).

We developed a new method for evaluating clinical symptoms of psoriasis during the observation period, by adding a parameter of time (the number of days) to the psoriasis area and severity index (PASI). This method was named the evaluation for prognosis with averaged PASI (E-PAP). Using this method, we assessed 14 cases to determine the following items: (1) clinical symptoms during the observation period; (2) clinical effects of treatments; (3) patient's satisfaction with treatments; and (4) necessity for additional and/or alternate treatments. We evaluated the usefulness of E-PAP in determining the prognosis of patients with psoriasis by comparing the uninterrupted monotherapy and intermittent therapy with cyclosporin A (Cys A). Although there were no differences in Cys A dosages after remission as well as adverse reactions between the two groups, the E-PAP value was significantly different between them. These results suggest that this method may be useful for determining the prognosis of patients with psoriasis.

Interaction of cultured keratinocytes and fibroblasts from human psoriatic and normal skin in immunodeficient mice.

Clarification of the pathogenesis of psoriasis requires separate studies of the epidermis, dermis, and inflammatory cells. We previously subcutaneously transplanted a mixture of cultured human keratinocytes and fibroblasts into mice to develop cysts with human skin structures. Using this method, we separately cultured psoriatic and normal keratinocytes and fibroblasts. Four mixtures were prepared: normal keratinocytes and normal fibroblasts (NK/NF); psoriatic keratinocytes and normal fibroblasts (PK/NF); normal keratinocytes and psoriatic fibroblasts (NK/PF); and psoriatic keratinocytes and psoriatic fibroblasts (PK/PF). Each mixture was transplanted into immunodeficient mice to observe formation of cysts and histological changes. The cysts varied in structure depending on the mixture, which suggests that psoriatic keratinocytes and fibroblasts had some abnormalities. Psoriatic fibroblasts may be partially responsible for thickening of the epidermis. Cell differentiation might have been accelerated in psoriatic keratinocytes after transplantation, resulting in the loss of epidermis structures.

Histological and immunocytochemical studies of human psoriatic lesions transplanted onto SCID mice.

To investigate the pathology of psoriasis, we developed an animal model for this disease using severe combined immunodeficiency (SCID) mice. These mice possess neither B nor T Lymphocytes so that both cellular and humoral immunities are impaired. For the in vivo study of psoriasis, human psoriatic skin was grafted on SCID mice. Long-term morphological and immunohistochemical changes in the grafted skin ware examined for up to 22 weeks after transplantation. The human skin graft were generally well maintained during this period, but the histological and immunohistochemical findings characteristic of psoriasis, except for acanthosis and hyperkeratosis, gradually disappeared as lymphocytic infiltration of the psoriatic lesions declined.

Expression of collagens I, II, X, and XI and aggrecan mRNAs by bovine growth plate chondrocytes in situ.

The cells responsible for skeletal growth are the chondrocytes of the cartilaginous growth plate. These cells differentiate through a series of maturational stages, establishing different zones in the growth plate. Among the major functions of these cells is the production of appropriate extracellular matrix, primarily composed of collagens and proteoglycans. To determine whether matrix synthesis varies with respect to maturational stage and in which cell populations different collagens are expressed, bovine growth plates were analyzed by in situ hybridization to mRNA and by Northern blot hybridization. The most abundant collagen mRNA in the growth plate was type-II collagen. This mRNA was present at relatively low levels in the most immature cells of the growth plate but increased several-fold as cells entered the proliferative stage and remained high through subsequent phases of maturation. Type-XI collagen mRNA and mRNA for the cartilage-characteristic proteoglycan, aggrecan, were codistributed with the type-II collagen mRNA; however, both were present in much smaller quantities. Type-X procollagen mRNA was localized to chondrocytes late in their maturation and was expressed at levels similar to the expression of type-II collagen. In situ hybridization of serial sections revealed that growth plate chondrocytes in their more mature stages contain both type-II and type-X collagen mRNA. Type-I collagen mRNA was not observed in growth plate chondrocytes at any maturational stage; rather, it was localized to a morphologically distinct population of cells attached to calcifying cartilage septa in the region of vascular invasion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin in psoriasis: continuous monotherapy versus intermittent long-term therapy.

Using two methods (continuous monotherapy and intermittent therapy) for the treatment of psoriasis with cyclosporine, we observed the clinical efficacy and adverse reactions of each treatment method for more than 36 months to evaluate the clinical usefulness of both methods. Thirty-seven cases were analyzed and the following results were obtained: 1) The PASI score evaluated at each visit was maintained between 5 and 10 by both treatment methods and the improvement rate was more than 70%, while there was no difference in the daily dose between the two treatment methods; 2) The period required to achieve remission tended to be prolonged by intermittent therapy, while no change was observed with continuous monotherapy; 3) The period up to relapse tended to become shorter with both treatment methods but this tendency was more marked with intermittent therapy; 4) E-PAP(evaluation for prognosis with averaged PASI) was lower in the continuous monotherapy group and the patients were more satisfied; 5) The incidence of adverse reactions was similar to that reported in previous studies, with no difference between the two treatment methods in this regard; 6) A significant increase in BUN levels was observed in elderly patients; 7) There were only three cases in which the drug was discontinued due to exacerbation and adverse reactions. Based on the above findings, continuous monotherapy seems to be of greater clinical usefulness than intermittent therapy.

Variant angina due to deficiency of intracellular magnesium.

A 51-year-old man was diagnosed as having variant angina by documentation of typical ST elevation during anginal attack and also by showing coronary arterial spasm (#2 and #12) during hyperventilation on coronary arteriography. Large quantities of calcium blocking agents and nitrates could not improve his symptoms. Lack of intracellular magnesium was suspected from a daily excretion of urine magnesium (5.3 mEq) and magnesium tolerance test (56.7%). After hourly infusion of magnesium sulfate (80 mEq), coronary spasm could not be induced by ergonovine.

Follow-up of clinical efficacy of iontophoresis therapy for postherpetic neuralgia (PHN).

A great variety of therapies have been attempted for PHN, including pharmacotherapy and physical therapy. However, there has been no decisive treatment, and reports of the clinical efficacy of all available therapies have been rather controversial. Almost all studies conducted so far have looked only at short-term therapeutic efficacy, and only a few investigators have conducted long-term observations or studies on long-term outcome. We followed up the clinical efficacy of iontophoresis therapy using lidocaine and methylprednisolone in 197 PHN patients. Monitoring conducted for an average of 4 years after completion of the treatment showed that pain remained unchanged or improved compared to pain observed upon completion of the treatment in 90.4% of patients. Although 42.6% of patients were still continuing some treatment, 90.9% were found to be able to take care of themselves. Findings obtained were reviewed and discussed from various viewpoints. Our findings showed that iontophoresis therapy is not only effective at the end of the treatment, but its efficacy is maintained over a long period of time, indicating that it is clinically very useful for the treatment of PHN.

HLA class I and II alleles and susceptibility to generalized pustular psoriasis: significant associations with HLA-Cw1 and HLA-DQB1*0303.

HLA alleles in generalized pustular psoriasis (GPP) were investigated to clarify the etiology and/or pathogenesis of this disease. Not only serological typing of HLA class I and II antigens but also genotyping of HLA class II alleles were carried out in twenty-six unrelated Japanese patients with GPP. These patients were classified according to their history of psoriasis vulgaris (PV). Serological typing revealed a significantly high incidence of HLA-Cw1 (Pc = 0.04) in the patients as compared with Japanese healthy controls. The frequency of HLA-B46 was particularly high in the patients with GPP and a previous history of PV. Genotyping of HLA class II alleles showed a highly significant increase in HLA-DQB1*0303 (Pc = 0.01) in the patients vs. the healthy controls. In particular, HLA-DQB1*0303 was significantly more frequent in the patients with no prior history of PV than in those with a history of PV. Analysis on linkage disequilibrium showed remarkably different patterns for HLA class II haplotypes between the patients and the healthy controls. Based on the comparative analysis among the amino acid sequences of the beta 1-domain of the HLA-DQB1*03 alleles, proline at residue 55 was suggested to be important as a common amino acid for determination of the susceptibility to GPP. These results revealed not only an association between the etiology and/or pathogenesis of GPP and HLA, but also different mechanisms of the immune response between the patients with GPP and PV.

[A successfully treated case of infective endocarditis due to Candida tropicalis].

A 34-year-old man, a heavy drinker, was admitted with a high fever and hematuria two months previously. Surgery was performed for acute sever pancreatitis and postoperatively antibiotics were administered with intravenous hyperalimentation. After discharge he was readmitted and infective endocarditis was strongly suspected because of high fever, hematuria, Osler's nodes, Janeway's lesions, splinter hemorrhages and mitral regurgitation. Penicillin G in combination with Gentamycine therapy was started on the first hospital day. On the second hospital day, blood culture revealed Candida tropicalis so Miconazole therapy was commenced. On the forth hospital day, he underwent surgery for replacement of a mitral prosthesis with a prosthetic valve because he had embolus in the radial artery. Despite intensive antifungal therapy, he showed no improvement in clinical symptoms. Then we changed the antifungal drug from Miconazole to Amphotericin B and 5-fluorocytosine. On the 109th hospital day, his clinical symptoms improved. Antifungal therapy was halted and at present 10 months later, he is healthy.