IL-12p40 is essential for the down-regulation of airway hyperresponsiveness in a mouse model of bronchial asthma with prolonged antigen exposure.

BACKGROUND: We previously reported a mouse model of bronchial asthma showing eosinophilic inflammation, but not airway hyperresponsiveness (AHR), after prolonged antigen exposure. This model showed an increase of IL-12 in the lung. OBJECTIVE: The aim of this study was to investigate the role of IL-12p40 in a murine asthma model with prolonged antigen exposures. METHODS: An ovalbumin (OVA)-induced asthma model was first established in wild-type (WT) and IL-12p40-deficient (IL-12p40(-/-)) mice. Both strains of mice were further exposed to either OVA (prolonged exposure group) or phosphate-buffered saline (positive control group) 3 days per week for 3 weeks. During week 4, both groups of mice were given a final challenge with OVA. RESULTS: Prolonged antigen exposures resulted in marked suppression of airway eosinophilia in both WT and IL-12p40(-/-) mice. However, AHR persisted in IL-12p40(-/-) but not in WT mice. There were no significant differences of IL-5, IL-13 or IFN-gamma levels in bronchoalveolar lavage fluid between WT and IL-12p40(-/-) mice. The hydroxyproline content of the lung and peribronchial fibrosis were, however, significantly increased in IL-12p40(-/-) mice. CONCLUSION: The results suggest that endogenous IL-12p40 is essential for inhibition of AHR and peribronchial fibrosis, but not eosinophilic inflammation, in a murine asthma model with prolonged antigen exposures.

Circulating KL-6/MUC1 as an independent predictor for disseminated intravascular coagulation in acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome (ARDS) patients show high levels of circulating mucin including KL-6/MUC1 (soluble MUC1 mucin). Because cancer mucin can bind vascular endothelial cells and platelets via selectins, mucin-selectin interactions are reported to trigger platelet aggregation and intravascular coagulation. Therefore, we hypothesized that KL-6/MUC1 is involved in the pathogenesis of disseminated intravascular coagulation (DIC) in ARDS. The aim of the current study is to evaluate the association between circulating KL-6/MUC1 and DIC in ARDS patients. DESIGN: Observational study with structured follow-up. SETTING: Intensive care unit in Hiroshima University Hospital. SUBJECTS: Fifty-six newly diagnosed patients with ARDS. INTERVENTIONS: Circulating levels of KL-6/MUC1 were measured during diagnosis and serially measured during the clinical course along with indices of respiratory failure, inflammation, coagulation and fibrinolysis and multiple organ dysfunction. RESULTS: Acute respiratory distress syndrome patients complicated with DIC showed significantly higher levels of serum KL-6/MUC1 than patients without DIC during the clinical course. Amongst the parameters analysed at diagnosis of ARDS, KL-6/MUC1 was an independent predictor for DIC complication. The baseline level of circulating KL-6/MUC1 at diagnosis of ARDS was significantly correlated with an increased DIC score following ARDS diagnosis. Using an optimum cutoff level of KL-6/MUC1 obtained by a receiver operating characteristic curve, the sensitivity and specificity for predicting future DIC development in ARDS patients were 88.9% and 55.3%, respectively. CONCLUSIONS: These results suggest that KL-6/MUC1 is associated with DIC development in ARDS patients. Elevated levels of KL-6/MUC1 at diagnosis could be a predictor of DIC complication in ARDS.

Increase of RP105-lacking activated B cells in the peripheral blood and salivary glands in patients with Sjögren's syndrome.

OBJECTIVE: To quantify the activated B cells in the peripheral blood and salivary glands of patients with Sjögren's syndrome (SS) by analyzing the expression of RP105 molecule on the B cells. METHODS: The expression of RP105 on the peripheral blood B cells of patients with SS (19 cases) was analyzed by flow cytometry. RP105-positive and negative B cells were sorted and cultured in vitro and the amount of immunoglobulins (IgG and IgM) produced in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Salivary gland biopsy samples from 9 SS patients were histologically evaluated and the sequential frozen sections were separately immunostained by anti-RP105 and anti-CD20 monoclonal antibodies. RESULTS: A significantly higher proportion of peripheral blood RP105-negative B cells was found in SS patients than in healthy individuals. RP105-negative, but not positive, B cells from SS patients were capable of producing IgG and IgM spontaneously in vitro, which was enhanced by the addition of Staphylococcus aureus Cowan I strain (SAC) or IL-6. Salivary glands from 2 of 9 SS patients were found to have lymphoid follicles whose germinal centers consisted of RP105-negative B cells. Moreover, a larger proportion of B cells extensively infiltrating the area other than lymphoid follicles was also RP105-negative. CONCLUSION: RP105-negative B cells, a subset of highly activated and well differentiated B cells, which are increased in number in the peripheral blood and extensively infiltrate salivary glands, may be responsible for the production of class-switched immunoglobulin in SS. In addition, those cells might be associated with the inflammation and tissue damage of the salivary glands.

The association of Behçet's disease with myelodysplastic syndrome in Japan: a review of the literature.

OBJECTIVE: [corrected] To determine the clinical characteristics of patients with myelodysplastic syndrome (MDS)-associated Behçet's disease (BD) in Japan. METHODS: 54 Japanese cases of MDS-associated BD obtained from the literature and from our own clinical experience were reviewed. The clinical features of MDS-associated BD were compared with those of the 1991 nationwide BD survey in Japan. RESULTS: In MDS-associated BD, the average age at onset was 42.6 years, which was 6.9 years later than for all BD patients; females developed disease more frequently than males (male: female ratio = 0.80). In MDS-associated BD cases, the occurrence of eye lesions was significantly lower, the frequency of intestinal lesions was markedly higher, and the rate of HLA-B51 positivity was lower than that in all BD. BD and MDS developed nearly simultaneously in 49.0% of cases; BD preceded MDS in 31.4% of the cases. The distribution of the age at BD onset showed two peaks, one in the 3rd decade and the other in the 6th decade. Females were more likely to develop younger-onset disease, while men were more likely to develop older-onset MDS-associated BD. Furthermore, in the older-onset group, BD was diagnosed together with or after the diagnosis of MDS, while half of the younger-onset group developed BD earlier than MDS. CONCLUSION: MDS-associated BD patients form a distinct subset of patients. There may, in fact, be two major groups of MDS-associated BD patients based on age, gender, and temporal relationship of the two diseases.

Efficient transplantation of human non-T-leukemia cells into nude mice and induction of complete regression of the transplanted distinct tumors by ricin A-chain conjugates of monoclonal antibodies SN5 and SN6.

In the present study, we established a dependable system by which human pre-B- and non-T/non-B-acute lymphoblastic leukemia (ALL) cells are efficiently transplanted into nude mice; the transplanted tumors provide a useful model for investigating the efficacy of antitumor agents in the in vivo therapy of human cancer. NALM-6 (a pre-B-ALL cell line) cells were transplanted under varying conditions as the pre-B-leukemia cells, whereas REH (a non-T/non-B-ALL cell line) cells were transplanted as the non-T/non-B-leukemia cells. Under optimal and near optimal conditions, 71 of 101 X-irradiated mice (70%) developed distinct tumors approximately 2 wk after i.d. inoculation of a mixture of NALM-6 cells and X-irradiated human fibrosarcoma cells. Under the same conditions, 9 of 11 mice (82%) developed tumors following i.d. inoculation of REH cells admixed with X-irradiated human fibrosarcoma cells. Examination of the tumor tissues demonstrated that the tumors are of leukemia origin but not of fibrosarcoma origin. To demonstrate the usefulness of the present tumors for investigating the efficacy of antitumor agents in the in vivo therapy of human cancer, immunotoxins were tested for their specific suppressive activity against growing tumors of the transplanted NALM-6 cells. To this end, monoclonal antibodies SN5 and SN6 which define a common ALL antigen, termed CALLA, and a novel leukemia-associated cell surface glycoprotein, termed gp160, respectively, were separately conjugated with the A-chain subunit of ricin, a plant toxin; CALLA and gp160 are expressed on the cell surface of various human non-T-leukemia cells including NALM-6 cells. The conjugates of SN5 and SN6 with ricin A-chain (RA) showed specific activity against the leukemia cells but not against control cells in an in vitro assay. To investigate their in vivo efficacy in suppressing tumor growth, nude mice which had been inoculated i.d. with NALM-6 cells 25 days in advance and bore distinct palpable tumors (5 to 6 mm in diameter) were divided into five groups. One group of mice was nontreated as a control. Each of the remaining four groups of mice was given an injection of one of the following agents: (a) purified control mouse IgG (IgG1); (b) purified antibodies SN5 (IgG1) and SN6 (IgG1); (c) control IgG-RA conjugate; or (d) SN5-RA and SN6-RA. Tumors in all mice of the first four groups including the untreated group grew continuously, causing the mice to die.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishment of ascitic tumor of human pre-B acute lymphoblastic leukemia in nonconditioned nude mice.

In the present study, an ascitic tumor of NALM-6, a human pre-B acute lymphoblastic leukemia cell line, was established in nude mice which had not been subjected to any preconditioning such as X-irradiation and/or splenectomy. The ascitic tumor was also established in X-irradiated mice. Under various conditions, the tumor transplantability was 100%. NALM-6 cells were initially inoculated i.p. into 8-day-old nude mice after being admixed with X-irradiated HT-1080, a human fibrosarcoma cell line. Then, the in vivo grown (for 10 wk) tumor cells were serially transplanted i.p. into X-irradiated adult (10 to 12 wk old) nude mice. After the serial passages, we succeeded in establishing a highly transplantable NALM-6 ascitic tumor in nonpreconditioned as well as X-irradiated nude mice without the addition of HT-1080 cells. The ascitic tumor cells do not lose transplantability after in vitro culture for 20 days. Cellular radioimmunoassay and fluorescence-activated cell sorter analysis indicated that the in vivo established NALM-6 tumor cells retained the antigenic phenotype of the parental NALM-6 cells. Titration experiments revealed a reciprocal relationship between survival time of the mice and the number of tumor cells inoculated. When appropriate numbers of tumor cells (e.g., 4 x 10(6) cells) were used for the inoculation, survival times of individual mice fell within a relatively narrow range; this narrow range will facilitate the experiments where the present tumor model is used for evaluating the in vivo efficacy of antitumor agents. The present ascitic tumor models, particularly the one established in nonpreconditioned nude mice, will be useful for evaluating the in vivo efficacy of anti-human leukemia agents as well as for studying the in vivo biological behavior of the transplanted leukemia cells.

Unique epitopes of common acute lymphoblastic leukemia antigen detected by new monoclonal antibodies.

In the present study we have generated four new monoclonal antibodies (mAbs), termed SN5, SN5a, SN5b, and SN5c, which are directed toward the human common acute lymphoblastic leukemia antigen (CALLA). SN5 and SN5c were generated separately by immunizing two mice with a leukemia antigen preparation isolated from uncultured non-T-/non-B-acute lymphoblastic leukemia cells whereas SN5a and SN5b were generated by immunizing a third mouse with intact KM-3 (a cultured non-T-/non-B-acute lymphoblastic leukemia cell line) cells. It was found that the binding activities of mAbs SN5 and SN5c generated by using an isolated leukemia antigen preparation were approximately twice as large as those of mAbs SN5a and SN5b generated by using intact leukemia cells. All four of the present mAbs induced antigenic modulation of CALLA on the leukemia cells in vitro; subclasses of mAbs appear to be an important factor which influences the kinetics of antigenic modulation. SN5, SN5a, and SN5c immunoprecipitated a distinct Mr 100,000 component from detergent-solubilized cell membrane antigens but SN5b failed to do so. These four mAbs together with J5, another anti-CALLA mAb, were individually tested in a solid phase radioimmunoassay for reactivity with the detergent extracts of various human tissues, i.e., kidney, lymph node, spleen, brain, liver, pancreas, lung, and heart. SN5, SN5a, SN5c, and J5 showed reaction only with kidney whereas SN5b did not show significant reaction with any tissues including kidney. However, SN5b as well as SN5 showed a significant reaction with kidney in an immunoperoxidase-staining test. These results indicate that the interaction of SN5b with a unique epitope on the CALLA moleucle is strongly disturbed by relatively mild detergents (deoxycholate, taurocholate, and Nonidet P-450). These detergents did not significantly disturb the reaction between other mAbs (SN5, SN5a, and SN5c) and the corresponding epitopes on the CALLA molecule. Competitive binding experiments show that the three epitopes recognized by SN5, SN5b, and SN5c are sufficiently close to each other to allow complete or nearly complete reciprocal inhibition of binding to CALLA present on leukemia cells. Peculiar inhibition patterns, however, were observed between SN5a and the other three mAbs. SN5, SN5b, and SN5c inhibited only partially the subsequent binding of SN5a to the leukemia cells. Conversely, SN5a inhibited nearly fully the subsequent bindings of SN5, SN5b, and SN5c. These results suggest another unique epitope defined by SN5a.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin.

In the present study, we developed an antitumor immunoconjugate that appears to be promising as a novel curative antitumor agent against a variety of human solid tumors. We generated a new antihuman endoglin (EDG) monoclonal antibody (mAb) K4-2C10 (or termed SN6f) that cross-reacts with mouse endothelial cells. Such cross-reactive anti-EDG mAbs have not been reported previously. This mAb was used to target tumor-associated vasculature in SCID mice inoculated with human tumors. No anti-EDG mAb or its immunoconjugates have previously been successfully used for targeting vasculature in vivo. In this study, MCF-7 human breast cancer cells were inoculated s.c. into SCID mice. K4-2C10 did not react with the MCF-7 cells but showed a weak reactivity with mouse endothelial cells. The mAb reacted with the proliferating endothelial cells more strongly than with the quiescent endothelial cells. The mAb exhibited much stronger reactivity (>10-fold) with human endothelial cells than with mouse endothelial cells and reacted strongly with vascular endothelium of tumor-associated blood vessels in a variety of human malignant tissues. Conjugates of K4-2C10 with ricin A chain (RA) and deglycosylated ricin A chain (dgRA) showed a weak but specific cytotoxic activity against murine endothelial cells in vitro; the 50% inhibitory dose of the RA and dgRA conjugates was 54 nm and 29 nm, respectively. Remarkable antitumor efficacy was observed when a small amount (a total of 60 microgram corresponding to 24% of the LD50 dose) of the dgRA conjugate was administered i.v. into SCID mice that had been inoculated s.c. with MCF-7. Unconjugated mAb K4-2C10 was not significantly effective in the inhibition of the tumor growth. The immunotoxin (IT) completely inhibited growth of the tumor in all of the treated mice (n = 8). Furthermore, similar antitumor efficacy was observed when the IT was administered i.v. into the tumor-inoculated SCID mice that had been pretreated with unconjugated K4-2C10 to block the potentially available weak binding sites of normal tissues. The strong therapeutic effects of the IT were reproduced in another set of therapeutic experiments. No significant side effects were observed in the mice. The differences in the tumor growth between the control group and the IT-treated groups were statistically significant. The IT showed antiangiogenic activity in the dorsal air sac method. The results indicate that K4-2C10 IT effectively treated the tumor-bearing mice by selectively inhibiting the tumor-associated blood vessels and by disrupting tumor-associated angiogenesis. The strong antitumor efficacy of the K4-2C10 IT is remarkable in view of the fact that K4-2C10 and its IT showed only a weak reactivity with mouse endothelial cells, and a relatively small amount of the IT was administered i.v. to treat s.c. tumors. We anticipate that the K4-2C10 IT will show much stronger antitumor efficacy and antiangiogenic activity in patients with solid tumors and other angiogenesis-associated diseases. The present results demonstrate for the first time that an anti-EDG mAb or its immunoconjugate can effectively target tumor-associated vasculature in vivo.

Association of serum endoglin with metastasis in patients with colorectal, breast, and other solid tumors, and suppressive effect of chemotherapy on the serum endoglin.

In this report, we present data indicating that the increased serum endoglin (EDG; CD105) quantitated by a double-antibody sandwich assay is associated with metastasis in patients with solid tumors including colorectal and breast carcinomas. In addition, we show that chemotherapy exerts a suppressive effect on the serum EDG. EDG is a proliferation-associated cell membrane antigen of human vascular endothelial cells. Furthermore, EDG is essential for angiogenesis. We generated two anti-EDG monoclonal antibodies (mAbs), termed SN6a and SN6h, defining different epitopes of EDG and developed a double-antibody sandwich assay to quantitate serum EDG in patients with solid tumors. SN6h possesses an exceedingly high antigen-binding avidity (K, 1.38 x 10(11) liters/mol), whereas SN6a possesses an ordinary avidity for a mAb directed to a cell surface antigen (K, 2.85 x 10(8) liters/mol). We measured serum samples from 101 patients with solid tumors (34 colorectal cancers, 16 breast cancers, and 51 other cancers), 8 patients with benign diseases, and 31 healthy volunteers. The serum level of EDG was significantly elevated in the patients with metastatic cancers. The mean serum EDG in the 42 metastasis-negative patients was 34.0 +/- 26.8 ng/ml (median value, 27.9 ng/ml), whereas the value in the 59 metastasis-positive patients was 63.8 +/- 72.5 ng/ml (median value, 37.2 ng/ml). The difference in EDG levels between the two groups was statistically significant (P = 0.012). Of the colorectal cancer patients, the difference in EDG levels between the 19 metastasis-negative patients and the 15 metastasis-positive patients was statistically significant (P = 0.02). In addition, the difference between the normal control (n = 31) and the 15 metastasis-positive colorectal cancer patients was statistically significant (P = 0.04). Of the breast cancer patients, the difference in EDG levels between the 11 metastasis-positive patients and the normal control was statistically significant (P < 0.005). In additional studies, we found that chemotherapy suppressed serum EDG levels in cancer patients. Of the 54 metastasis-positive patients with solid tumors, the mean serum EDG in the 32 chemotherapy-receiving [chemotherapy(+)] patients was 44.7 +/- 41.9 ng/ml (median value, 36.1 ng/ml), whereas the value in the 22 chemotherapy(-) patients was 102.4 +/- 99.5 ng/ml (median value, 64.8 ng/ml). The difference in serum EDG between the two groups is statistically significant (P < 0.005). In the majority of metastasis-positive patients who were not receiving chemotherapy, serum EDG was elevated. The results suggest that serum EDG may be a useful marker for monitoring early signs of metastasis and cancer relapse in a long-term follow-up of solid tumor patients.

Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies.

Endoglin (EDG, CD105) is a proliferation-associated antigen on endothelial cells. In this study, two new anti-EDG monoclonal antibodies (mAbs) Y4-2F1 (or termed SN6j) and P3-2G8 (SN6k) were generated and used for treating distinct preformed tumors. These mAbs, both IgG1-kappa antibodies, cross-reacted weakly with mouse endothelial cells but defined epitopes different from the epitope defined by a previously reported anti-EDG mAb K4-2C10 (B. K. Seon et al., Clin. Cancer Res., 3: 1031-1044, 1997). SN6j and SN6k reacted strongly with human endothelial cells and vascular endothelium of malignant human tissues but showed no significant reactivity with tumor cells per se. The deglycosylated ricin A chain (dgRA) conjugates of the two mAbs showed a weak but specific cytotoxic activity against murine endothelial cells in vitro. In the therapeutic studies, severe combined immunodeficient mice were inoculated s.c. with MCF-7 human breast cancer cells and left untreated until palpable tumors of distinct size (4-6 mm in diameter) appeared. Mice with the distinct tumors were treated by i.v. administration of individual anti-EDG conjugates, unconjugated mAbs, or a control conjugate. Long-lasting complete regression of the tumors was induced in the majority of tumor-bearing mice (n = 8 for each conjugate) when 40 microg of the individual conjugates were administered three times via the tail vein. It is remarkable that the tumors remained regressed without further therapy for as long as the mice were followed (i.e., 100 days). Control conjugate did not induce regression of the tumors in any of the treated mice, although weak nonspecific effects were observed in some of the mice (n = 8). The effects of unconjugated mAbs were small with the dose used, i.e., 34 microg three times. The anti-EDG conjugates showed antiangiogenic activity in the dorsal air sac assay in mice. The results suggest good potential of these conjugates for the clinical application.

New monoclonal antibodies SN3, SN3a, and SN3b directed to sialic acid of glycoprotein on human non-T leukemia cells.

We have generated and characterized three new monoclonal antibodies (mAbs), termed SN3, SN3a, and SN3b, which are directed to sialic acid of a glycoprotein(s) on human non-T leukemia cells. These mAbs were generated by immunizing mice with an antigen preparation isolated from cell-membrane glycoconjugates of NALM-1, a pre-B leukemia cell line. The initial characterization of the mAbs consisted of a sensitive cellular radioimmunoassay against various cultured human leukemia-lymphoma (HLL) and nonmalignant cell lines. They strongly reacted with all four (all three in the case of SN3a) non-T/non-B HLL cell lines tested and both pre-B HLL cell lines tested. However, they reacted with only one of three B HLL cell lines tested. In addition, these mAbs did not react with other cell lines, which include T- and myelomonocytic HLL cell lines and nonmalignant B-cell lines. Normal peripheral blood cells were also tested; the mAbs reacted with B cells and granulocytes but not with T cells, monocytes, erythrocytes, or platelets. In a test using SN3 and SN3b with uncultured cell specimens derived from various cancer patients, the mAbs primarily reacted with non-T/non-B and B HLL specimens, as well as with chronic myelocytic leukemia specimens. The biochemical nature of antigenic determinants defined by the three mAbs was studied by treating the non-T leukemia cells with sialidase and proteases. The results show that the antigenic determinants defined by these mAbs all contain a sialic acid residue(s) that is attached to the cells via a protein backbone(s). Competitive binding experiments show that binding of SN3 to the leukemia cells was blocked almost completely by SN3a and SN3b, as well as by BA-1. Both SN3 and SN3a are IgG1 antibodies, whereas SN3b is an IgM antibody; SN3b showed a strong complement-mediated cytotoxic activity against non-T leukemia cells.

Reactivation of HSV-1 in primates by transcorneal iontophoresis of adrenergic agents.

Transcorneal iontophoresis of adrenergic agents has been shown to reactivate latent herpes simplex virus type 1 (HSV-1) and to produce viral shedding in the tear film in rabbits and mice, but not, to date, in nonhuman primates. In this study, we demonstrated induced reactivation of latent HSV-1, viral shedding, and production of ocular lesions in nine squirrel monkeys (Saimiri sciureus) by iontophoresis of 6-hydroxydopamine (6-HD) with topical instillation of epinephrine or with iontophoresis of timolol. Monkey corneas were scarified and inoculated with McKrae strain HSV-1 on three separate occasions. All monkeys received daily intramuscular prednisolone for 39 or 46 days prior to ionotophoresis. Once latency was established, a single ionotphoresis of 6-HD was performed on both eyes in five of the monkeys, followed by 1% epinephrine given topically four times daily for 5 days. Iontophoresis of timolol was performed on both eyes in the other four monkeys once per day on 3 consecutive days. Eight of the ten eyes receiving 6-HD and epinephrine shed HSV-1; seven eyes developed deep punctate, dendritic, or geographic corneal lesions. Seven of the eight eyes receiving timolol shed HSV-1; six of the eyes developed lesions suggestive of HSV-1 specific corneal lesions. The methods used in this report were slightly different from those used to reactivate HSV-1 in rabbits, in that repeated inoculations with HSV-1 and repeated intramuscular injections with prednisolone were required; however, these results demonstrate that iontophoresis of adrenergic agents can produce shedding and recurrent epithelial lesions in the nonhuman primate.

Recurrent HSV-1 corneal epithelial lesions induced by timolol iontophoresis in latently infected rabbits.

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent corneal epithelial lesions were assessed following ocular iontophoresis of 0.01% timolol at 0.8 mAmp for 8 min for 3 consecutive days in 17 rabbits latently infected with HSV-1 strain McKrae. The collection of ocular tear film for the detection of HSV-1 ocular shedding and the slit lamp biomicroscopic evaluation for HSV-1 epithelial lesions began on day 4 after the first iontophoresis and continued for 7 consecutive days. The tear film was collected on a Dacron swab with care being taken to avoid swabbing the corneal epithelium. The observed HSV-1 lesions were characterized as deep punctate lesions, dendritic lesions and geographic epithelial defects. The ratio of total days of eyes exhibiting HSV-1 epithelial lesions to the total number of observation days was 113/235 (48%). There were 46 punctate lesions, 27 dendritic lesions and 40 geographic epithelial defects. The ratio of positive swabs to total swabs was 77/235 (33%). Of the 113 positive lesion days, 65 (58%) were associated with a positive swab. Of the 77 positive swabs, 65 (84%) were associated with an epithelial lesion. Of the 122 negative lesion days, 110 (90%) were associated with a negative swab. Of the 158 negative swabs, 110 (70%) were associated with no epithelial lesions. By chi-square analysis, there was a significant association between HSV-1 swabs and HSV-1 lesions (P less than 0.001). These results demonstrate that ocular iontophoresis of timolol induces both HSV-1 ocular shedding and recurrent HSV-1 corneal epithelial lesions in rabbits latently infected with HSV-1 strain McKraw.

Corneal nerves are necessary for adrenergic reactivation of ocular herpes.

Herpes simplex virus type 1 (HSV-1) can infect the cornea and achieve ganglionic latency. HSV-1 can later be activated by a variety of effectors although the exact mechanism of reactivation is unknown. Rabbits harboring latent HSV-1 strain McKrae can be induced to shed virus by ocular iontophoresis of epinephrine to the cornea. No studies have been done to investigate if corneal nerves are necessary for epinephrine induction of HSV-1 ocular shedding. We did penetrating keratoplasty (PKP) in one eye each of 23 rabbits; the other eye served as an unoperated control. The surgery effectively denervates the area of the transplant for up to 90 days. Eighteen rabbits carrying latent HSV-1 strain McKrae received corneas from uninfected rabbits. Five uninfected rabbits with no latent virus received corneas from rabbits harboring latent HSV-1. On days 10-14 after penetrating keratoplasty, 24 eyes in the HSV-1 latent group and all ten uninfected eyes received iontophoresis of 0.01% epinephrine (0.8 mAmps for 8 min or 0.6 mAmps for 6 min) once daily for 3 days by means of an eye cup whose diameter was less than the diameter of the transplant. Six rabbits in the HSV-1 latent group received intravenous injections of cyclophosphamide (75 mg/kg) and dexamethasone (4 mg/kg). Following iontophoretic or immunosuppressive induction, the eyes were swabbed daily for 9 days. Of the 12 rabbits with latent virus which were treated by iontophoresis, one of the transplanted eyes and eight of the nontransplanted eyes were induced to shed virus. The mean duration of shedding in the nontransplanted eyes was 3.25 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Trifluridine decreases ocular HSV-1 recovery, but not herpetic lesions after timolol iontophoresis.

To determine the effect of a topically applied antiviral agent on shedding of herpes simplex virus type 1 (HSV-1) into the tear film and corneal epithelial lesions, ten rabbits latently infected with HSV-1 were subjected to transcorneal iontophoresis of 0.01% timolol once a day for 3 consecutive days to induce viral shedding and lesions. Iontophoretic induction was performed similarly in five uninfected rabbits as controls. Half of the infected rabbits and all of the uninfected controls received topical 1.0% trifluridine five time a day for 9 days, beginning the day after the first iontophoresis. All eyes were examined daily for 10 days by slit-lamp biomicroscopy and tear film samples collected on swabs were analyzed for virus. In the infected rabbits, the eyes treated with trifluridine had significantly fewer swabs positive for HSV-1 than the untreated eyes (P less than 0.001); however, there was no significant difference in the numbers of lesions in the treated and untreated eyes. The uninfected controls had no positive swabs and developed no lesions. These results suggest that topical treatment with trifluridine may reduce recovery of HSV-1 from the tear film, but does not affect the incidence of iontophoretically induced corneal epithelial lesions.

Recurrent HSV-1 corneal lesions in rabbits induced by cyclophosphamide and dexamethasone.

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent corneal epithelial lesions were assessed following an intravenous injection of cyclophosphamide (75 mg/kg) and 24 hr later an intravenous injection of dexamethasone (4 mg/kg) in 24 eyes of 15 rabbits latently infected with HSV-1 strain McKrae. Sampling for HSV-1 ocular shedding and epithelial lesion began on the day after cyclophosphamide injection and continued for 8 consecutive days. Ocular tear film was collected on a Dacron swab with care taken to avoid swabbing the corneal epithelium. Slit-lamp biomicroscopic examination was used to observe and characterize induced HSV-1 corneal epithelial lesions as deep punctate keratitis, dendritic keratitis or geographic epithelial defects. The ratio of positive days of epithelial lesions per total days was 82/187 (44%). There were 32 deep punctate lesions, 17 dendritic lesions, and 33 geographic epithelial defects. The ratio of positive swabs per total swabs was 78/187 (42%). Of the 82 positive lesion days, 54 (66%) were associated with a positive swab. Of the 78 positive swabs, 54 (69%) were associated with an epithelial lesion. Of the 54 days of both positive lesion and swab, 16 (30%) were associated with a dendritic lesion. By chi-square analysis, there was a significant association between HSV-1 swabs and HSV-1 lesions (P less than 0.001). These results confirm that intravenous injections of cyclophosphamide and dexamethasone induce both HSV-1 ocular shedding and recurrent herpes simplex corneal lesions in rabbits latently infected with HSV-1 strain McKrae.

Timolol induces HSV-1 ocular shedding in the latently infected rabbit.

Timolol iontophoresis into the eye can induce herpes simplex virus type 1 (HSV-1) shedding in rabbits latently infected with HSV-1 strain McKrae. Anodal iontophoresis of 0.01% timolol was done at 0.8 mAmp for 8 min once a day for 3 consecutive days. Viral shedding was determined by the presence of HSV-1 in the preocular tear film obtained by eye swabs. In two experiments, iontophoresis of 0.01% timolol resulted in all eyes (18/18) shedding HSV-1 for an average duration of 4.3 days. When 5.0% timolol was applied topically to rabbit eyes supersensitized by iontophoresis of 6-hydroxydopamine (6-HD), all eyes (10/10) shed virus for an average duration of 2.9 days. All eyes (12/12) receiving iontophoresis of 6-HD, pre- and posttreatment with topical application of 5.0% timolol, and posttreatment with topical application of 1.0% epinephrine shed virus for an average duration of 3.6 days. Eyes treated with topical application of 5.0% timolol alone showed no difference in HSV-1 ocular shedding, compared with untreated eyes. We concluded that both iontophoresis of 0.01% timolol and topical application of 5.0% timolol to adrenergically supersensitized eyes induced HSV-1 shedding reliably and with a high frequency, and that topically applied timolol does not block the HSV-1 ocular shedding induced by epinephrine in adrenergically supersensitized eyes.

In vitro and in vivo cytotoxic activity of anti-human leukemia monoclonal antibodies SN5c and SN6 daunorubicin conjugates.

Murine monoclonal antibodies SN5c specific for the common acute lymphoblastic leukemia antigen (CALLA) and SN6 specific for a novel GP160 tumor associated antigen expressed on non-T ALL and myelomonocytic leukemia cells were conjugated to daunorubicin via an intermediate dextran carrier. The resulting monoclonal antibody-daunorubicin conjugates retained the immunoreactivity of the unlabeled antibody to antigen positive leukemia target cells. In addition, these conjugates demonstrated selective cytotoxic activity when tested against a panel of human leukemia cell lines and/or human leukemia patient samples of peripheral blood or bone marrow origin. The SN5c and SN6-daunorubicin immunoconjugates were superior to a non-specific isotype matched MOPC-daunorubicin conjugate in in vitro cytotoxicity assays. Free daunorubicin, however, was more cytotoxic than either immunoconjugate but lacked selectivity. SN5c-daunorubicin and SN6-daunorubicin combined were as effective as free daunorubicin when used for in vivo therapy and led to complete ablation of established NALM-6 tumors in an athymic nude mouse model. The SN5c-daunorubicin conjugate was also shown to be significantly less toxic than free daunorubicin in non-tumor bearing Balb/c mice. These studies indicate that mAb-daunorubicin conjugates can be constructed which retain specific binding and exhibit selective cytotoxicity against human leukemia cells and suggest that they may have therapeutic applications.

Activation of platelets in bronchial asthma.

STUDY OBJECTIVES: To investigate whether platelets are activated in asthmatics with increased release of preformed mediators and to investigate the influence of oral administration of theophylline on them. DESIGN: Comparison of the intracellular free calcium concentration ([Ca2+]i) in platelets as an indicator of platelet activation, CD62P expression on platelets, and the chemokine regulated upon activation in normal T cells expressed and presumably secreted (RANTES) level in platelet-rich buffer supernatants between asthmatics and normal subjects. SETTING: The respiratory outpatient clinics, Hiroshima University, Japan. PARTICIPANTS: Twenty-five normal volunteers, 19 asthmatics taking no oral drugs associated with asthma treatment (group A), and 18 asthmatics taking oral theophylline (group B). MEASUREMENTS AND RESULTS: While the resting [Ca2+]is in platelets were similar among the three groups, the [Ca2+]is in group A were significantly higher than those in normal subjects (p<0.05) and group B (p<0.01) after thrombin or 9,11-epithia-11,12-methano-thromboxane A2 (STA2) stimulation in the absence of external Ca2+. The CD62P expression level and RANTES level in group A after STA2 stimulation were significantly higher than those in normal subjects and group B (p<0.05). CONCLUSIONS: We conclude that agonist-mediated activation of platelets is augmented in asthmatics resulting in enhanced release of chemokine such as RANTES, which could be suppressed by oral administration of theophylline.

Anomalous origin of the left coronary artery from the pulmonary artery. New operative technique.

We report a new operative technique for repair of anomalous origin of the left coronary artery from the pulmonary artery. The principles of the proposed technique are left main coronary angioplasty using a transected main pulmonary artery, side-to-side anastomosis of the aorta and newly created left coronary artery, and direct anastomosis of the transected pulmonary artery. No prosthetic material is used in this procedure. Our experience in two adults (a 35-year-old man and a 68-year-old woman) indicated that this technique permits two coronary system repair for any anatomic variation of the left coronary artery without the use of prosthetic material. This is more advantageous in infants.