Inactivation of foodborne pathogenic and spoilage micro-organisms using ultraviolet-A light in combination with ferulic acid.

The low energy of UV-A (315-400 nm) is insufficient for disinfection. To improve UV-A disinfection technology, we evaluated the effect of ferulic acid (FA) addition on disinfection by UV-A light-emitting diode (LED) (350-385 nm) against various food spoilers and pathogens (seven bacteria and four fungi species). Photoantimicrobial assays were performed at FA concentrations below the MIC. The MIC of the isomerized FA, consisting of 93% cis-form and 7% trans-form, was very similar to that of the commercially available FA (trans-form). Irradiation with UV-A (1·0 J cm-2 ) in the presence of 100 mg l-1 FA resulted in enhanced reducing of all of the tested bacterial strains. A combination of UV-A (10 J cm-2 ) and 1000 mg l-1 FA resulted in enhanced reducing of Saccharomyces cerevisiae and one of the tested filamentous fungi. These results demonstrated that the combination of a short-term application of UV-A and FA at a low concentration yielded synergistic enhancement of antimicrobial activity, especially against bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial contamination is one of the most serious problems for foods, fruit and sugar thick juices. UV light is suitable for the nonthermal decontamination of food products by inactivating the contaminating micro-organisms. However, UV-A exposure is insufficient for disinfection. This study demonstrates that the combination of UV-A LED light (350-385 nm), which is not hazardous to human eyes and skin, and ferulic acid (FA), a known phytochemical and food additive, provides synergistic antimicrobial activity against foodborne pathogenic and spoilage micro-organisms. Therefore, FA addition to UV-A light treatment may be useful for improvement of UV-A disinfection technology to prevent food deterioration.

Onychomycosis caused by Fusarium proliferatum.

Fusarium infections in humans are usually opportunistic, but the fungus sometimes infects healthy persons, causing keratomycosis or onychomycosis. Onychomycosis is usually caused by F. solani or F. oxysporum. We report the first two cases of onychomycosis caused by F. proliferatum, and discuss methods of diagnosis and effective treatment. Nail samples from the two patients were examined by direct microscopy, cultured, and identified morphologically and genetically as F. proliferatum. Both patients were treated successfully with oral itraconazole, even though the minimum inhibitory concentration of itraconazole was relatively high in Patient 1. This is the first report of F. proliferatum as an agent of onychomycosis. Itraconazole may be effective in the treatment of onychomycosis caused by F. proliferatum.

The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc.

Reduction in klotho gene expression causes accelerated senescence in klotho mutant mice. We have now found two key substances, phosphorus and zinc, which affect the appearance of klotho phenotypes. Klotho mutant homozygotes fed nonpurified diet with a phosphorus concentration of 1.03 g/100 g showed typical klotho phenotypes. However, most of the klotho phenotypes no longer appeared in male homozygotes fed a 0.4 g/100 g phosphorus diet. These homozygotes were capable of spermatogenesis. In the kidneys of the rescued male homozygotes, klotho protein expression was clearly detected. On the other hand, female klotho mice required supplementation of 0.25 g/100 g zinc orotate to the 0.4 g/100 g phosphorus diet to be rescued. Unlike in the rescued male mice, klotho protein levels in the kidneys of the rescued females were quite low. Wild-type (C3H/He) mice fed 1.5 or 1.0 g/100 g phosphorus diets had lower klotho protein expression in the kidneys than those fed a 0.4 g/100 g phosphorus diet (Kruskal-Wallis test, P < 0.05). These results indicate that dietary phosphorus and zinc modulate the phenotypes of klotho mice, and that klotho expression in the kidneys is regulated not only in klotho mutant mice, but also in wild-type mice.

Topical application of cyclosporin A induces rapid-remodeling of damaged anagen hair follicles produced in cyclophosphamide administered mice.

Adult C3H mice which had either anagen IV or anagen VI hair follicles were given the anti-tumor drug cyclophosphamide, and cyclosporin A or minoxidil were topically applied to the mice daily from the 4th day after cyclophosphamide administration. In the mice that had anagen IV-hair follicles, 0.5% cyclosporin A induced very thick and long hairs after 21 days of cyclophosphamide administration, while vehicle and 1% minoxidil induced sparsely visible, short hairs. In the mice which received cyclosporin A, the injured hair follicles seemed to remodel themselves into intact anagen hair follicles and restart the production of hairs, instead of shifting to telogen. In the mice that had anagen VI-hair follicles at the time of cyclophosphamide administration, complete alopecia occurred within the first 7 days in all groups. After 14 days of cyclophosphamide administration, hair regrowth was observed in both the 0.5% cyclosporin A-group and the 1% minoxidil- group with the predominant effect over the vehicle. This study shows that anagen hair follicles respond to cyclophosphamide in different ways depending on their stages (IV and VI), and that the damaged anagen IV hair follicles have the potential of remodeling themselves, which is promoted by topical cyclosporin A administration.

Establishment of enzyme immunoassay for measuring serum sultopride levels.

Measurement of serum sultopride levels was performed using an enzyme immunoassay. Little or no cross-reactivity with metabolites of sultopride and other drugs was found. The results of reproducibility, recovery, and dilution testing were all good enough for clinical use. A comparison between the measurement values of this method (y) with that of high-performance liquid chromatography (x) showed high correlation (n = 211, r = 0.991, p < 0.0001, y = 0.99x + 107.5). In a comparison between the sultopride dose and serum levels in 161 patients, interindividual differences were large (19 times for same doses), implying that the serum level cannot be predicted from the dosage. The method was found to be reliable for serum level measurements of sultopride and useful for monitoring compliance and assessing the optimal dose.

KF19418, a new compound for hair growth promotion in vitro and in vivo mouse models.

KF19418, a newly synthesized compound, stimulated proliferation of cultured hair bulb cells from new born mice in concentration-dependent manner in the range under 10 microM. In the culture system of whole skin pieces from 4-week-old mice which we earlier established, KF19418 promoted hair follicle elongation as in the case of minoxidil. After topical application for 2 weeks of KF19418 or minoxidil to dorsal skin of hair-clipped mouse alopecia model, KF19418 at 1% suspension accelerated hair regrowth at a rate comparable to 1% minoxidil solution. Thus, it was shown that KF19418 directly stimulated hair follicle in vitro and had hair growth promoting activities in vivo.

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice.

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.

[Assessment on intermittent intravenous cyclophosphamide pulse therapy in diffuse proliferative lupus nephritis].

OBJECTIVE: To determine whether intravenous cyclophosphamide pulse therapy (IVCY) is effective for treating patients with diffuse proliferative lupus nephritis (DPLN) who were 1) refractory to methylprednisolone pulse therapy (MP) or 2) could not be treated with MP because of severe diabetes or steroid induced psychosis. METHODS: Seven patients with biopsy proven DPLN were studied after informed consent. Five of them received IVCY after a failure to achieve renal remission with at least 2 cycles of MP therapy. Of the other 2 patients, one had severe diabetes and the other a history of steroid induced psychosis. Bolus therapy with cyclophosphamide (0.5 g/m2 body surface area) was given once a month for 6 consecutive months and then once every 3 months for a total treatment period of 1 year. All patients were given oral prednisone, 0.5 mg/kg per day. The prednisone dose was tapered to the minimal dose required for controlling the disease. After 1 year, the renal status of the patients were evaluated. RESULTS: At 1 year, 4 of the 7 patients achieved substantial improvement. Although the other 3 patients did not satisfy the definition of substantial improvement, none of them had progressive disease. Adverse events were mild and did not require any treatment, with 2 cases of leukocytopenia without fever or major infection. No cases of hemorrhagic cystitis or amenorrhea were observed. CONCLUSIONS: IVCY was 1) effective in the treatment of DPLN which was refractory to MP and 2) relatively safe with minimal side effects.

IL-12-encoding plasmid has a beneficial effect on spontaneous autoimmune disease in MRL/MP-lpr/lpr mice.

Systemic lupus erythematosus (SLE) is characterized by immune abnormalities explained by the overproduction of Th(2)cytokines such as autoantibody production and polyclonal B cell activation. We examined the effect of administering a DNA plasmid encoding IL-12 on the lupus-like disease of MRL/MP-lpr/lpr (MRL/lpr) mice. Treatments were delivered intramuscularly every 4 weeks, starting at 4 weeks of age. This intervention significantly inhibited the accumulation of CD4(-)CD8(-)T cells, and reduced lymphadenopathy and splenomegaly. A significant decrease in serum IgG anti-DNA autoantibody titers was observed, and plasmid IL-12 therapy was also associated with a reduction in the proteinuria and glomerulonephritis characteristic of this disease. Serum IFN-gamma level was increased by inoculating IL-12 encoding plasmid, suggesting that the cytokine balance was skewed towards Th(1). The clinical implications of this suppression of autoimmune disease are also discussed.

Severity of seropositive isolated Raynaud's phenomenon is associated with serological profile.

OBJECTIVE: To examine the relationship between the clinical severity of seropositive isolated Raynaud's phenomenon (RP) and its serological background by analyzing digital blood flow data obtained by laser Doppler flowmetry (LDF). METHODS: We analyzed digital blood flow by LDF in 13 healthy volunteers, 55 patients with seropositive isolated RP, and 13 patients with anti-Scl-70 antibody positive systemic sclerosis (SCL). The serological profiles of patients with RP were as follows: 30 patients had the anti-centromere antibody (C) and 19 the anti-RNP antibody (RNP). We designated the RP in each patient group as C-RP, RNP-RP, and SCL-RP. We used an "arm-raising test" by which blood pressure could be passively depressed, and the cold provocation test, which induced vasoconstriction through the sympathetic reflex. We defined 2 variables, the recovery velocity after cold exposure (RV-CE) and the increase in the amplitude of the digital pulse wave during the arm-raising test (IA-AR), that are the most reliable and sensitive variables indicating the severity of RP. RESULTS: Both RV-CE and IA-AR correlated significantly with the clinical severity of RP. In IA-AR and RV-CE, there was a significant difference between C-RP and RNP-RP (IA-AR 107.1 +/- 25.63 vs 37.4 +/- 17.25%; RV-CE 0.0667 +/- 0.010 vs 0.035 +/- 0.0096 V/s), showing that C-RP tended to be less severe than RNP-RP. CONCLUSION: We defined 2 variables that correlated with the clinical severity of RP; using them we found that anti-centromere antibody positive RP is less severe than RNP-RP

[Hematological abnormalities of primary Sjogren's syndrome].

Sjogren's syndrome (SS) is an autoimmune disease characterized by a chronic inflammatory response mainly localized to the lacrimal and salivary glands. However, it sometimes involves extraglandular organs culminating in systemic disorders. Hematological abnormalities are not uncommon, although they rarely have clinical significance. In this study we examined 99 patients with primary SS who visited our hospital during 1989 to 1999. Patient's mean age was 54.1 years and 95 out of 99 were female. Lymphopenia and leukopenia was noted in 35 patients (35.3%) and 26 patients (26.2%) respectively, and 7 patients (7.1%) had thrombocytopenia. 43 patients (43.4%) had either of these hematological abnormalities. Patients with lymphopenia showed significantly low frequency of arthralgia and anti-SS-A/B antibody was more common in this group. Only one patient in this group required prednisolone therapy because of polyarthritis and general fatigue while others needed no specific therapy. Patients with thrombocytopenia were significantly younger and a male/female ratio was higher than those without this abnormality. They had higher tendency to accompany with skin eruption, positive anti-SS-B antibody, anti-nuclear antibody and rheumatoid factor. Three out of 8 patients with thrombocytopenia were treated with prednisolone according to the protocol for idiopathic thrombocytopenic purpura. All of 3 patients had positive PA-IgG and normocellular bone marrow. Autoimmune mechanism such as polyclonal B cell activation may play a role in the pathogenesis of thrombocytopenia.

Several selective protein kinase C inhibitors including procyanidins promote hair growth.

We have previously reported that procyanidin oligomers selectively promote growth of murine hair epithelial cells in vitro and stimulate anagen induction in vivo. We report here the possible relationship between the protein kinase C-inhibiting activity of procyanidins and their hair-growing activity. Of the procyanidins, procyanidin B-2 and procyanidin C-1, which selectively inhibit protein kinase C, intensively promote hair epithelial cell proliferation in vitro and stimulate anagen induction in vivo. On the other hand, procyanidins, which inhibit both protein kinase C and A, showed relatively low activity in in vitro and in vivo evaluations. We also found that calphostin C, which is a selective inhibitor of protein kinase C, possesses hair epithelial cell growth-promoting activity in vitro and anagen phase-inducing hair-growing activity in vivo. Other selective protein kinase C inhibitors, such as hexadecylphosphocholine, palmitoyl-DL-carnitine chloride, and polymyxin B sulfate, also show marked anagen phase-inducing hair-growing activity in vivo. Nonselective protein kinase inhibitors, such as staurosporine and K252a, inhibit the growth of hair epithelial cells. 1,2-Dioctanoyl-sn-glycerol, a protein kinase C activator, dose-dependently decreases the growth of hair epithelial cells. Forskolin, an adenylate cyclase activator, promotes hair epithelial cell growth and boosts the growth-promoting effect of procyanidin B-2. It is speculated that the hair-growing activity of procyanidins is related to their protein kinase C-inhibiting activity.

Establishment of the anti-Klotho monoclonal antibodies and detection of Klotho protein in kidneys.

A novel gene, klotho (kl), which is involved in the development of a syndrome resembling human aging in mice, was recently identified. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. There also exists a splice variant of kl mRNA which encodes a putative secreted protein in both human and mouse. In this study, to characterize the physiological roles of Klotho protein, we established three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG(2)a), KM2119 (rat IgG(2)b), and KM2365 (mouse IgG(1)). In Western blots, KM2076 and KM2119 specifically recognized a 130 kDa Klotho protein in the mouse and human kidney membrane fractions. To detect the human Klotho protein, the sandwich-type ELISA system with KM2076 and KM2365 was established. Using the ELISA system, we detected the human Klotho protein as low as 20 ng/ml in the supernatant of Chinese hamster ovary cells (CHO cells), introduced the human klotho gene. KM2076 and KM2119 specifically gave a positive staining by immunohistochemical staining in paraffin or frozen sections of the kidneys from wild-type mice but not in those from kl mice. Strong staining was observed especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction pattern in the paraffin sections of rat and human kidneys. The mAbs established in this paper will serve as useful analytical, pathological, and diagnostic tools to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging.

Dendritic cells are associated with augmentation of antigen sensitization by influenza A virus infection in mice.

To study the mechanisms responsible for enhanced sensitization of inhaled antigen in respiratory viral infections, we examined the contribution of dendritic cells (DC) and T lymphocytes to the development of inhalation sensitization during infection with influenza A virus in mice. BALB/c mice were sensitized by inhalation of ovalbumin (OA) from 3 to 7 days after the inoculation with influenza A virus, and were challenged with OA 3 weeks later. Airway responsiveness and serum OA-specific IgE were increased. The numbers of eosinophils and CD4(+) and CD8(+) T cells in bronchoalveolar lavage fluid were also increased. These changes were not observed in animals only sensitized with OA or only inoculated with the virus. In animals only inoculated with the virus, DC were immunohistochemically detected on the bronchial epithelium on days 2-5. With OA inhalation after virus inoculation, DC with high expression of MHC class II were retained for 5 weeks. These results show that influenza virus infection induces the migration of DC to the bronchial epithelium, and that simultaneous inhalation of antigen causes the loading of antigen-peptide / class II molecule complex on DC. Thus, the migration of DC in viral infection may play some role in the augmentation of antigen sensitization.

[Two cases of acute lupus peritonitis].

We report two cases of systemic lupus erythematosus (SLE) diagnosed when acute peritonitis was appeared. Case 1 was a 20 year-old woman suffering from stomachache and right lower abdominal pain. Case 2 was a 40 year-old woman with diarrhea, epigastralgia, pollakisuria. In both cases, their peritoneal fluids were exudative with positive autoantibodies. After high dose steroid therapy, abdominal symptoms and ascites improved promptly. However, due to the complication of lupus nephritis, additional therapy was necessary. To characterize the feature of lupus peritonitis (LP), we examined the clinical and laboratory findings of LP from the literature. In patients with acute LP, abdominal pain, vomiting, diarrhea were significantly more common compared with chronic LP patients (P < 0.05), and fever, arthritis, central nervous system involvement and cystitis were more common. In patients with chronic LP, pleural effusion and pericardial effusion were more common compared with acute LP patients. Gastrointestinal manifestations such as abdominal pain, vomiting and diarrhea were more common in patients with acute LP compared with patients with chronic LP. Most patients with chronic LP were asymptomatic, ascites and serositis being the only clinical findings. The response to steroid therapy was better in acute LP.

[Two cases of HIV infection accompanied with borderline personality disorder].

We report here two cases of HIV infection with a borderline personality disorder. Case 1 was a 25-year-old male patient who was diagnosed with HIV infection 4 years ago. Borderline personality disorder was also diagnosed at that time. Although he was referred to our hospital in 1999, we had to refer him to another hospital for his regular outpatient hemodialysis. Case 2 was a 24-year-old male patient who had borderline personality disorder since 1996. He was diagnosed with HIV infection in 1999 and referred to our hospital. He ignored rules in visiting clinics such as prior reservations and frequently called doctors, case-workers and nurses. After several visit he intentionally took excessive sedative medicines and called a case-workers at our hospital. He was admitted to our hospital for three days. After he was discharged, we set limitations for his behavior not to harm himself and to obey the rules in visiting clinics. In other countries investigators report that borderline personality disorder is more common in HIV-infected persons. It may be because persons with borderline personality disorder are more likely to engage in high-risk sexual behavior, which is also applicable to these two cases. As HIV infection is rapidly prevailing in Japan, it is possible that the chance are that this disorder will be seen more frequently in HIV infected cases.

Immunomodulatory effect of a plasmid expressing CD40 ligand on DNA vaccination against human immunodeficiency virus type-1.

CD40 ligand is a costimulatory molecule which acts a potent immunomodulator. We found the mice inoculated with human CD40 ligand expression plasmid (pMEhCD40L) combined with human immunodeficiency virus type-1 (HIV-1) DNA vaccine exhibited both humoral and cellular antigen-specific immunological enhancement. The expression of hCD40L induced predominantly antigen-specific immunoglobulin G (IgG) antibody response while it failed to induce mucosal IgA response. Delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) activity were induced in a dose-dependent manner. Examination of the relative levels of the two IgG subclasses showed that co-injection of pMEhCD40L enhanced IgG2a response without suppressing IgG1 response. Similarly, the expression of pMEhCD40L enhanced not only T helper 1 (Th1)- but also Th2-type cytokine production. In conclusion, co-inoculation of pMEhCD40L with DNA vaccine was shown to be a useful way to enhance CTL responses without suppressing the humoral immune response in acquired immune deficiency syndrome (AIDS) patients.

[Successful treatment of primary CNS lymphoma diagnosed by 201thallium-single photon emission computed tomography (201Tl-SPECT) with whole-brain radiation therapy in an AIDS patient].

The patient, a 51-year-old male with a two year history of AIDS, was admitted to our hospital because of hemiparalysis and vomiting. The MRI study showed multiple lesions with ring-enhancement in the right basal brain area. Empirical therapy for toxoplasma encephalitis was started. After 64 days, the subsequent brain MRI showed deterioration. A 201Tl-SPECT study was performed and the findings were consistent with those of malignant lymphoma (ML). The patient was treated with 40 Gy of whole brain radiation, MRI showed partial response to this therapy, and clinical improvement was achieved. The definitive diagnosis of primary CNS lymphoma can be made only by brain biopsy, and many cases have been diagnosed at autopsy. The clinical and radiological findings of primary CNS lymphoma resemble toxoplasma encephalitis. An empirical therapy for toxoplasma encephalitis is recommended to avoid brain biopsy in these cases. The use of 201Tl-SPECT for the differential diagnosis of these diseases have been reported. Considering the poor prognosis of primary CNS lymphoma in AIDS, the application of 201Tl-SPECT before empirical therapy for toxoplasma must be important for appropriate treatment.