Functional characterization of recombinant snake venom rhodocytin: rhodocytin mutant blocks CLEC-2/podoplanin-dependent platelet aggregation and lung metastasis.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and ß-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. SUMMARY: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and ß-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTßWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTßWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTßK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

A case of exfoliative esophagitis with pemphigus vulgaris.

Autoimmune blistering skin diseases, including pemphigus vulgaris, rarely involve the esophagus. We report a case of exfoliative esophagitis with pemphigus vulgaris. A sloughing esophageal cast observed by endoscopy was dissected esophageal squamous epithelium in all layers. Our case is the fifth case of pemphigus vulgaris associated with esophageal cast formation recorded in the medical literature. Prednisolone was administered, and both the pemphigus vulgaris and exfoliative esophagitis improved. Upon findings of exfoliative esophagitis by endoscopic examination, we should consider the coexistence of blistering skin diseases, including pemphigus vulgaris.

Inhibition of acid production in dental plaque bacteria by green tea catechins.

The inhibition of acid production from dental plaque and mutans streptococci by epigallocatechin gallate (EGCg), one of the green tea catechins, was examined. The effect of EGCg solution on dental plaque pH was investigated. Subjects rinsed their mouths with 2 mg/ml EGCg solution and then, after 30-min interval, rinsed their mouths with 10% sucrose. Plaque samples were collected at appropriate times and the pH was measured. The pH values of plaque samples from 15 volunteers were significantly higher after treatment with catechin than after treatment with water. EGCg inhibited pH fall when cariogenic bacteria grown in medium with or without sucrose were incubated with sugar. In medium without sucrose, cultured cells were killed time-dependently by EGCg treatment. However, EGCg did not kill cells cultured in medium containing sucrose. Also, EGCg did not kill oral streptococci adhering to a saliva-coated hydroxyapatite disk. EGCg and epicatechin gallate inhibited lactate dehydrogenase activity much more efficiently than epigallocatechin, epicatechin, catechin or gallocatechin. These results suggest that EGCg is effective in reducing acid production in dental plaque and mutans streptococci.

Attempts to transmit porcine reproductive and respiratory syndrome virus by aerosols under controlled field conditions.

An experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) was established in 150 five-month-old pigs housed in a fan-ventilated finishing facility, the infected barn. To determine whether air exhausted from the wall fans contained infectious PRRSV, a trailer containing 10 four-week-old PRRSV-naive sentinel pigs was placed 10 m from the building from day 3 after the 150 pigs were infected until day 10. To connect the two airspaces, one end of an opaque plastic tube, 15 m in length and 5 cm in diameter, was fastened to the wall fan of the infected barn, and the other end was placed inside the trailer. Air from the building was exhausted into the trailer 24 hours a day for seven consecutive days and PRRSV infection was monitored in the infected pigs and the sentinel pigs. Air samples were collected from the infected barn and the trailer. PRRSV infection was detected in the infected pigs three and seven days after they were infected, but not in the sentinel pigs. All the air samples were negative for PRRSV by PCR, virus isolation and a pig bioassay.

Studies on the carriage and transmission of porcine reproductive and respiratory syndrome virus by individual houseflies (Musca domestica).

The objectives of the study were to determine the site of porcine reproductive and respiratory syndrome virus (PRRSV) in individual houseflies, to assess whether an individual housefly could transmit PRRSV to a susceptible pig, and to compare the ability of PCR, virus isolation and a pig bioassay to detect PRRSV in houseflies. In the first experiment 26 houseflies were fed on a pig infected experimentally with PRRSV; 13 were processed as a whole fly homogenate, while an exterior surface wash and a gut homogenate were collected from the other 13. Infectious PRRSV was recovered from nine of the whole fly homogenates, 12 of the gut homogenates and one of the exterior surface washes. In the second experiment, two of 10 individual houseflies, which had fed on an infected pig, transmitted PRRSV to a susceptible pig in a controlled manual transmission protocol. In the third experiment, single flies or pools of 30 flies were immersed in different concentrations of a PRRSV inoculum, then tested by PCR, virus isolation and bioassay. The virus was detected at a concentration of 10(1) TCID50/ml by PCR, 10(2) TCID50/ml by the bioassay and 10(3) TCID50/ml by virus isolation.

Transmission of porcine reproductive and respiratory syndrome virus by houseflies (Musca domestica).

Three hundred houseflies were allowed to feed on donor pigs viraemic with porcine reproductive and respiratory syndrome virus (PRRSV) on the fifth, sixth and seventh days after the pigs had been inoculated with the virus. After 60 seconds, the flies' feeding was interrupted, and they were transferred manually to feed to repletion on a naive recipient pig housed in a separate room. To enhance the chance of the flies obtaining the pigs' blood, the back of each pig was scarified with sandpaper until a slight haemorrhage was visible. The PRRSV was transmitted from the donor to the recipient pigs, and PRRSV RNA was detected by reverse transcriptase-PCR from homogenates of the flies. In a second experiment, 210 houseflies were allowed to feed to repletion on a PRRSV-infected pig on the sixth day after it had been inoculated, and were then maintained under laboratory conditions. Groups of 30 flies were collected immediately after they had fed and six, 12, 24, 48, 72 and 96 hours later, and were tested for PRRSV. Homogenates of the flies collected up to six hours after feeding were PCR- and pig bioassay-positive, but the others were negative by both tests.

Evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under controlled field conditions.

The aim of this study was to determine whether porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted by aerosol under field conditions. A total of 210 five-month-old PRRSV-negative pigs were housed in a mechanically ventilated finishing facility containing 11 pens. Pen 1 contained 10 pigs (indirect contact controls) and pen 2 remained empty, providing a barrier of 2.5 m from the remaining pigs in pens 3 to 11. Fifteen or 16 of the pigs in each of pens 3 to 11 were infected experimentally with a field isolate of PRRSV and the other six or seven pigs served as direct contact controls. Five days after the pigs were infected, two trailers containing 10 five-week-old PRRSV-naive sentinel pigs were placed along each side of the building; one was placed 1 m from the exhaust fans on one side of the building, and the other was placed 30 m from the fans on the other side, and the sentinel pigs remained in the trailers for 72 hours. They were then moved to separate buildings on the same site, 30 and 80 m, respectively, from the infected barn, and their PRRSV status was monitored for 21 days. The direct and indirect contact control pigs became infected with PRRSV but the sentinel pigs did not.

Muscular sarcoidosis.

There are four types of muscular sarcoidosis: nodular, chronic myopathy, acute myositis, and asymptomatic. The nodular type is important because it may be confused with a soft tissue tumor. Magnetic resonance (MR) imaging provides specific findings. A star-shaped central structure of decreased signal intensity, a "dark star" sign, is seen on the axial images. A long nodule with an inner stripe of decreased signal intensity and outer stripes of increased signal intensity, a "three stripes" sign, is seen on the coronal and sagittal images. In the chronic myopathy type, the role of MR imaging is limited. Gallium scintigraphy, which reflects activity of inflammation, may show increased uptake. In the acute myositis type, MR imaging shows increased signal intensity, and gallium scintigraphy shows increased uptake; however, these findings are nonspecific.

[The study of house monitoring system in Welfare Techno-House Mizusawa].

In 1999, 8 elderly people aged over 70 stayed for 5 days in Mizusawa Welfare Techno-house and their health conditions and activities of daily life were monitored using 16 sensors attached around the house. To determine a few feasible, practical sensors and optimal sensor positions, accumulated data was analyzed. Because of bud get conditions only two sensors (infrared sensor and life line monitor sensor) were placed in one voluntary house and the data were transferred from the voluntary house to the techno-house through the ISDN and CATV lines. The data were analyzed every days. Only 2 sensors to detect were placed and attached this year, however one more sensor to detect physical conditions such as ECG records during sleeping time, will be used next year. On the other hand, an integrate system that can analyze many data transferred from various sensors simultaneously was developed. To introduce this system into privatehouse, many problems such as privacy protection, security, etc must be solved.

Genetically manipulated bacterial toxin as a new generation mucosal adjuvant.

Cholera toxin (CT) and heat-labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co-administered protein antigen (Ag). Both LT and CT induce B7-2 expression on antigen-presenting cells (APCs) for subsequent co-stimulatory signalling to CD4+ T cells. CT directly affects CD4+ T cells activated via the TCR-CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)-4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL-4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4+ Th2 cells, which provided effective help for the Ag-specific mucosal immunoglobulin (Ig)A, as well as serum IgG1, IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7-2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4+ T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.

Protective immunity to Streptococcus mutans induced by nasal vaccination with surface protein antigen and mutant cholera toxin adjuvant.

In this study, mice were immunized nasally with surface protein antigen of Streptococcus mutans serotype c (PAc) and a nontoxic A subunit mutant of cholera toxin (mCT) E112K, as a mucosal adjuvant. Immunization with PAc and mCT elicited significant PAc-specific secretory IgA in saliva and in nasal secretions. Antibody-forming cell (AFC) analysis confirmed the antibody (Ab) titers by revealing significant numbers of PAc-specific IgA AFCs in the submandibular gland and nasal passages. Furthermore, CD4(+) T cells from cervical lymph nodes exhibited significant proliferative responses when restimulated with PAc in vitro. Importantly, mice that were nasally immunized with PAc plus mCT E112K exhibited significantly reduced oral colonization by S. mutans. These results show that nasal administration of PAc and mCT E112K is potentially an effective mucosal vaccine against dental caries and reduces the colonization of S. mutans in the oral cavity.

Identification of genetically diverse sequences (ORF 5) of porcine reproductive and respiratory syndrome virus in a swine herd.

The ability of genetically diverse strains of porcine reproductive and respiratory syndrome virus (PRRSV) to coexist in a 1750-sow farm was assessed through the case study describing a chronically infected farm, and also by an animal experiment involving the use of swine bioassay. The case study employed a program of monitoring sera from suckling, nursery, and finishing pigs for the presence of PRRSV by polymerase chain reaction (PCR) and virus isolation (VI). The swine bioassay tested homogenates, consisting of lymphoid and pulmonary tissues, collected from 60 breeding animals from the same farm. The open reading frame (ORF) 5 portion of selected positive PRRSV detected from sera or tissues were nucleic acid sequenced and their phylogenies compared. The results indicated the presence of 3 genetically diverse groups, designated PRRSV-A, -B, and -C. Sequence heterology ranged from 5.8 to 11% between groups. Sequence homology ranged from 98.7 to 99.8% within groups. Swine bioassay verified the presence of PRRSV-A in 1 of 60 animals, and no evidence of strains B or C were detected. This paper indicates that based on the evaluation of ORF 5, genetically diverse strains of PRRSV appear to coexist, although the frequency and significance of this observation is not understood.

Transmission of porcine reproductive and respiratory syndrome virus from persistently infected sows to contact controls.

The objective of this study was to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could persist in non-pregnant sows and if persistently infected sows could transmit virus to naive contact controls. Twelve PRRSV-naive, non-pregnant sows (index sows) were infected with a field isolate of PRRSV and housed in individual isolation rooms for 42 to 56 days postinfection. Following this period, 1 naive contact sow was placed in each room divided by a gate allowing nose-to-nose contact with a single index sow. Index sows were not viremic at the time of contact sow entry. Virus nucleic acid was detected by polymerase chain reaction, and infectious virus was detected by virus isolation in sera from 3 of the 12 contact sows at 49, 56, and 86 days postinfection. All 3 infected contacts developed PRRSV antibodies. Virus nucleic acid was detected in tissues of all of the 12 index sows at 72 or 86 days postinfection. Nucleic acid sequencing indicated that representative samples from index and infected contacts were homologous (> 99%) to the PRRSV used to infect index sows at the onset of the study. This study demonstrates that PRRSV can persist in sows and that persistently infected sows can transmit virus to naive contact animals.

Gene transfer of calcitonin gene-related peptide prevents vasoconstriction after subarachnoid hemorrhage.

We sought to determine whether adenovirus-mediated gene transfer in vivo of calcitonin gene-related peptide (CGRP), a potent vasodilator, ameliorates cerebral vasoconstriction after experimental subarachnoid hemorrhage (SAH). Arterial blood was injected into the cisterna magna of rabbits to mimic SAH 5 days after injection of AdRSVCGRP (8x10(8) pfu), AdRSVbetagal (control virus), or vehicle. After injection of AdRSVCGRP, there was a 400-fold increase in CGRP in cerebrospinal fluid. Contraction of the basilar artery to serotonin in vitro was greater in rabbits after SAH than after injection of artificial cerebrospinal fluid (P<0.001). Contraction to serotonin was less in rabbits with SAH after AdRSVCGRP than after AdRSVbetagal or vehicle (P:<0.02). Basal diameter of the basilar artery before SAH (measured with digital subtraction angiogram) was 13% greater in rabbits treated with AdRSVCGRP than in rabbits treated with vehicle or AdRSVbetagal (P:<0.005). In rabbits treated with vehicle or AdRSVbetagal, arterial diameter after SAH was 25+/-3% smaller than before SAH (P<0.0005). In rabbits treated with AdRSVCGRP, arterial diameter was similar before and after SAH and was reduced by 19+/-3% (P<0.01) after intracisternal injection of CGRP-(8-37) (0.5 nmol/kg), a CGRP(1) receptor antagonist. To determine whether gene transfer of CGRP after SAH may prevent cerebral vasoconstriction, we constructed a virus with a cytomegalovirus (CMV) promoter, which results in rapid expression of the transgene product. Treatment of rabbits with AdCMVCGRP after experimental SAH prevented constriction of the basilar artery 2 days after SAH. Thus, gene transfer of CGRP prevents cerebral vasoconstriction in vivo after experimental SAH.

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract.

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.

The spatial arrangement of L and M cones in the peripheral human retina.

The spatial arrangement of L and M cones in the human peripheral retina was estimated from red-green color naming of small test flashes (0.86 min of arc, 555 nm, constant intensity) presented at different locations (grid with 1.5 min of arc steps) centered at 17 degrees temporal eccentricity. Simulated red-green color naming ratings were generated by a model based on an ideal observer for all possible patterns of placement and relative numerosities of L and M cones, constrained by the anatomical data on the statistics of cone spacing at this retinal location. The best matching simulated performance as compared to the human observer's data determined the cone array most likely to produce that observer's color naming results. The mosaics for two color normal observers showed L and M cones randomly arrayed over this retinal region. Consequences of random cone placements for spectral sampling and color opponency are discussed.