Increased severity of local and systemic anaphylactic reactions in gp49B1-deficient mice.

gp49B1 is an immunoglobulin (Ig) superfamily member that inhibits FcstraightepsilonRI-induced mast cell activation when the two receptors are coligated with antibodies in vitro. The critical question of in vivo function of gp49B1 is now addressed in gene-disrupted mice. gp49B1-deficient mice exhibited a significantly increased sensitivity to IgE-dependent passive cutaneous anaphylaxis as assessed by greater tissue swelling and mast cell degranulation in situ. Importantly, by the same criteria, the absence of gp49B1 also resulted in a lower threshold for antigen challenge in active cutaneous anaphylaxis, in which the antigen-specific antibody levels were comparable in gp49B1-deficient and sufficient mice. Moreover, the absence of gp49B1 resulted in a significantly greater and faster death rate in active systemic anaphylaxis. These results indicate that gp49B1 innately dampens adaptive immediate hypersensitivity responses by suppressing mast cell activation in vivo. In addition, this study provides a new concept and target for regulation of allergic disease susceptibility and severity.

Phenotypic changes of bone marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are genetically deficient in mast cells.

The ability of mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) to generate serosal mast cells (SMC) in vivo after adoptive transfer to mast cell-deficient mice has been defined by chemical and immunochemical criteria. BMMC differentiated and grown from WBB6F1-+/+ mouse progenitor cells in medium containing PWM/splenocyte-conditioned medium synthesized a approximately 350,000 Mr protease-resistant proteoglycan bearing approximately 55,000 Mr glycosaminoglycans, as defined by gel filtration of each. Approximately 85% of the glycosaminoglycans bound to the cell-associated BMMC proteoglycans were chondroitin sulfates based upon their susceptibility to chondroitinase ABC digestion; HPLC of the chondroitinase ABC-generated unsaturated disaccharides revealed these glycosaminoglycans to be chondroitin sulfate E. As determined by heparinase and nitrous acid degradations, approximately 10% of the glycosaminoglycans bound to BMMC proteoglycans were heparin. In contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient WBB6F1-W/Wv mice 15 wk after intraperitoneal injection of BMMC synthesized approximately 650,000 Mr protease-resistant proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 105,000 Mr. Thus, after adoptive transfer, the SMC of the previously mast cell-deficient mice were like those recovered from the normal WBB6F1-+/+ mice that were shown to synthesize approximately 600,000 Mr proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 115,000 Mr. As assessed by indirect immunofluorescence staining and flow cytometry using the B1.1 rat mAb (an antibody that recognizes an epitope located on the neutral glycosphingolipid globopentaosylceramide), approximately 5% of BMMC bound the antibody detectably, whereas approximately 72% of the SMC that were harvested from mast cell-deficient mice 15 wk after adoptive transfer of BMMC were B1.1-positive; approximately 82% of SMC from WBB6F1-+/+ mice bound the antibody. These biochemical and immunochemical data are consistent with the results of previous adoptive transfer studies that characterized mast cells primarily on the basis of morphologic and histochemical criteria. Thus, IL-3-dependent BMMC developed in vitro, cells that resemble mucosal mast cells, can give rise in vivo to SMC that express phenotypic characteristics of connective tissue mast cells.

SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells.

SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.

Mast cell activation enhances airway responsiveness to methacholine in the mouse.

Mast cell-deficient mutant mice and their normal littermates were used to determine whether activation of mast cells by anti-IgE enhances airway responsiveness to bronchoactive agonists in vivo. Pulmonary conductance was used as an index of airway response as the mice were challenged with increasing intravenous doses of methacholine (Mch) or 5-hydroxytryptamine (5-HT). Mast cell activation with anti-IgE enhanced pulmonary responsiveness to Mch in both types of normal mice (P < 0.0001 by analysis of variance) but not in either genotype of mast cell-deficient mouse. Additionally, anti-IgE pretreatment of genetically mast cell-deficient W/Wv mice whose mast cell deficiency had been repaired by infusion of freshly obtained bone marrow cells or bone marrow-derived cultured mast cells from congenic normal mice led to significant (P < 0.0001) enhancement of Mch responsiveness. 5-HT responsiveness was not significantly influenced by anti-IgE pretreatment in any of the mice studied. The data support the hypothesis that IgE-mediated activation of mast cells enhances pulmonary responsiveness to cholinergic stimulation.

Effect of lucanthone hydrochloride on the radiation response of intestine and bone marrow of the Chinese hamster.

A sublethal dose of 100 mg lucanthone hydrochioride/kg (Miracil D, Nilodin; NSC-14574) administered ip into Chinese hamsters [median lethal dose for 30-day survival (LD50/30) of 315 mg/kg] reduced the radiation tolerance of the small intestine and had little or no effect on the radiation tolerance of the bone marrow. Lucanthone hydrochloride was administered at various times before and after whole-body 60Co gamma-irradiation. The median lethal dose for 7-day survival (LD50/7), indicative of death from gastrointestinal epithelial denudation, was reduced from 1,235 rads to minimum values of 995 rads or 985 rads by lucanthone hydrochloride inoculation 10 hours before irradiation or 7.5 hours post irradiation, respectively. The LD50/30, indicative of death from bone marrow stem cell depletion, remained unaltered at approximately 990 rads over the entire treatment scheme, which indicated that the radioresponsiveness of bone marrow stem cells was unaffected by lucanthone hydrochloride. The lucanthone hydrochloride effect was reversible in that control values of LD50/7 were attained by 40 hours post inoculation. Serum concentration of lucanthone hydrochloride in the Chinese hamster, determined spectrophotometrically, reached a peak of 8 microgram/ml by 1.5 hours post inoculation and then decreased exponentially with a half-life of approximately 6 hours, so that by 30 hours post inoculation it was unmeasurable.

The effect of resection on local failure in irradiated non-oat cell carcinoma of the lung.

From January 1969 through December 1979, 171 patients completed a course of high dose definitive radiotherapy alone for non-oat cell carcinoma of the lung. During the same period, 53 patients completed a course of definitive postoperative radiotherapy after undergoing resection of the primary tumor. The two groups were otherwise very similar with regard to patient related and tumor related variables. A detailed analysis of the incidence of clinically documented local (in-field) failure on the basis of clinical T and N stages was performed. A comparison of the incidence of local failure as the first site of failure for patients with T1-2 tumors demonstrated a statistically significant decrease in local failure in patients whose primary tumors were resected. This was true for all patients with T1-2 tumors whose failure status was known (p less than .001), for all patients known to have failed (p less than .001), and for patients whose clinical node status was other than gross mediastinal (N2) lymphadenopathy (p less than .05). Local failure was decreased in patients with clinical T3 tumors who underwent resection, but the difference was not significant (p greater than .1). Histology (epidermoid vs. non-epidermoid) had no apparent effect on the frequency of local failure, either with or without resection. A review of past experience indicates that local failure is common after definitive irradiation alone, and is due to a low rate of sterilization of the primary tumor, even with tolerance doses of irradiation. Data are presented to support a reappraisal of the role of combined resection and irradiation in future clinical trials, to reduce the present unacceptably high rate of local failure in potentially curable patients treated by irradiation alone.

Protective effects of viscous solutions in phacoemulsification and traumatic lens implantation.

We compared the endothelial protection offered by 1% hyaluronate sodium (Healon), 3% hyaluronate sodium and 4% chondroitin sulfate (Viscoat), and a nonviscous irrigating solution (BSS Plus) during phacoemulsification with and without traumatic intraocular lens implantation. Vital-dye staining and scanning electron microscopy were used to determine acute damage to rabbit corneal endothelium. Cell damage during phacoemulsification alone was not significantly different from that in unoperated controls (12.5%). Cell damage after traumatic lens insertion was significantly greater in the groups treated with BSS Plus (76.2%) and Healon (41.4%) than in either paired Viscoat-treated group (21.1% and 17.4%, respectively). Viscoat (but not Healon) was noted to be adherent to the cornea at the end of the procedure in one third of the cases, indicating that Viscoat remains in the anterior chamber during surgery. We attribute this to chondroitin sulfate's newtonian characteristics, allowing it to maintain viscosity in the face of high flow rates.

Molecular and Cellular Biology of Rodent Mast Cells.

Mouse bone marrow-derived mast cells (BMMC) are progenitors for mucosal mast cells (MMC) and connective tissue mast cells (CTMC). These populations differ in their expression of granule mediators and surface markers. Molecular cloning of most of the components of mast cell granules has provided an essential tool for understanding mast cell heterogeneity and development. Cytokine regulation of mast cell protease gene expression has facilitated the in vitro characterization of the pathways of mast cell maturation towards the CTMC and MMC phenotypes.

Ciprofloxacin-resistant bacterial keratitis.

Ciprofloxacin, a new broad-spectrum antibiotic effective against a variety of gram-positive and gram-negative bacteria, has recently become available in topical ophthalmic solution (3 mg/ml) for the treatment of bacterial keratitis. It has rapidly become the drug of choice in treating bacterial keratitis. We treated three patients with bacterial corneal ulcers that were resistant to ciprofloxacin, yet were effectively treated with other topical antimicrobial agents. The initial culture results are important in the therapy of corneal ulcers.

Complications of contact lens wear after radial keratotomy in an animal model.

In 12 rabbits radial keratotomy was performed on one eye with no treatment to the contralateral eye. Cellulose acetate butyrate contact lenses were fitted bilaterally for extended wear. The eyes were monitored weakly for three weeks with a standardized slit-lamp grading method. Corneal neovascularization occurred earlier and progressed further in eyes that had radial keratotomy than in the control eyes (P less than .0025). These results suggested that patients who require contact lenses after radial keratotomy may be at higher risk for complications such as corneal neovascularization.

Comparison of topical ciprofloxacin to conventional antibiotic therapy in the treatment of ulcerative keratitis.

We evaluated the efficacy of ciprofloxacin (3 mg/ml) as the sole topical antibiotic used to treat infectious keratitis in 14 patients. We compared the ciprofloxacin-treated group to a retrospective control group of 30 consecutive culture-positive patients treated with conventional therapy in which cefazolin (50 mg/ml) and fortified gentamicin sulfate (9.1 mg/ml) solutions were used. We found no remarkable difference between the control group and the ciprofloxacin-treated group regarding patient age, risk factors, need for hospitalization, and virulence of organism isolated. The average time to healing in culture-positive ciprofloxacin-treated patients was 34 +/- 33 days vs 45 +/- 71 days in the control group and this difference was not statistically significant. The duration of antibiotic therapy in the culture-positive ciprofloxacin-treated group was 27 +/- 15 days vs 33 +/- 50 days in the control group. Four of the 30 control patients required modification of their antibiotic regimen, whereas no ciprofloxacin-treated patient required a change. Ciprofloxacin appears to be an effective single agent in the treatment of ulcerative keratitis.

Nd:YAG laser photo-induced adhesion of the corneal epithelium.

We retrospectively studied eight patients with recurrent corneal erosions treated with the Nd:YAG laser using 0.4- to 0.5-mJ pulses applied to the region of Bowman's layer through an intact epithelium. All eight patients had resolution of their symptoms after treatment. Mean follow-up time was 21.2 months (range, 12.6 to 36.6 months). A patient who was scheduled for diagnostic enucleation for a posterior choroidal mass consented to undergo this laser treatment with varied energy settings six days before his enucleation. His cornea was studied with specular microscopy, light microscopy, and transmission electron microscopy. Light microscopy of the cornea disclosed rare 100-microns defects in Bowman's layer with subjacent compaction of the anterior stromal lamellae. Electron microscopy showed minute foci of disruption in Bowman's layer with new collagen formation. Fine fibrils connected the basal epithelial cells to the new collagen. Nd:YAG laser photo-induced adhesion of the corneal epithelium may represent an effective treatment alternative for patients with recurrent corneal erosions.

Late partial dislocation of a laser in situ keratomileusis flap.

We report an apparently atraumatic asymptomatic flap dislocation 4 months after uneventful laser in situ keratomileusis (LASIK) in the right eye of a 43-year-old woman. The patient developed partial dislocation of the LASIK flap during the week after the 4 month examination. The LASIK flap was subsequently lifted to perform an enhancement, and the postenhancement course has been unremarkable. This case illustrates the potential susceptibility of LASIK flaps to dislocation either spontaneously or, more likely, after presumed minor trauma as late as 4 months after the original procedure.

Intraoperative corneal pachymetry in eyes having radial keratotomy.

We prospectively studied 45 eyes that had radial keratotomy for correction of myopia to determine whether significant changes in corneal thickness occurred during the surgical procedure and which paracentral corneal region was the thinnest consistently. We used a standard bidirectional technique with a diamond knife. The inferotemporal paracentral region was the thinnest most frequently (38% of eyes) both pre-incision and post-incision. However, each of the other paracentral regions measured the thinnest in a smaller percentage of eyes: temporal (28% pre-incision and post-incision); inferior (19% pre-incision, 21% post-incision); nasal (11% pre-incision, 9% post-incision); superior (4% pre-incision and post-incision). A statistically significant reduction in corneal thickness occurred intraoperatively in all regions.

Effect of topical ciprofloxacin 0.3% and ofloxacin 0.3% on the reduction of bacterial flora on the human conjunctiva.

PURPOSE: To evaluate quantitatively over time the reduction in bacterial flora on the human conjunctiva after treatment with topical ciprofloxacin 0.3% (Ciloxan) or topical ofloxacin 0.3% (Ocuflox). SETTING: Sinai Hospital of Baltimore, Baltimore, Maryland, USA. METHODS: Three study arms each consisted of 20 culture-positive eyes from patients 55 years or older. Pretreatment cultures were performed in all eyes. Eyes in the ciprofloxacin and ofloxacin arms received 1 antibiotic drop every 5 minutes for 3 doses. The conjunctiva of each treatment eye was recultured 15, 30, 60, and 120 minutes after application of the final antibiotic drop. Eyes in the control arm were recultured at corresponding time points. After 48 hours of incubation, colony counts were performed. Data were transformed into log units, and statistical analysis was performed. When compared to no treatment, instillation of ofloxacin 0.3% did not produce a significant reduction in bacterial colony forming units (CFUs) at 15, 30, or 60 minutes (P =.17). A marginally significant reduction was achieved 120 minutes after administration (P =.051). RESULTS: When compared to no treatment, instillation of ciprofloxacin 0.3% produced a significant reduction in bacterial CFUs at 15 minutes; this effect persisted for at least 2 hours (P <.0001). The reduction in bacterial CFUs by ciprofloxacin was significantly greater than that by ofloxacin at all measurements (P <.0001). CONCLUSIONS: Ciprofloxacin 0.3% markedly reduced bacterial flora on the ocular surface within 15 minutes of instillation, and the effect lasted for at least 2 hours.

Pseudomonas cepacia keratitis.

Pseudomonas cepacia has recently become recognized as a virulent pathogen responsible for nosocomial infections in hosts with altered immunity. It has been implicated in endophthalmitis and conjunctivitis, and is resistant to conventional antipseudomonal therapy. No cases of P. cepacia keratitis have been reported in the literature. We report such a case in association with topical steroid and contact lens use following penetrating keratoplasty. In addition, we developed an experimental model of P. cepacia keratitis in the rabbit. P. cepacia should be considered as a cause of infectious keratitis especially in nosocomial infections in immunocompromised corneas.

Inhibition of contact lens-induced corneal neovascularization in radial keratotomized rabbit eyes.

Cellulose acetate butyrate contact lenses were fitted for extended wear on 28 rabbits 6 weeks after radial keratotomy. Fourteen rabbits received 0.03% flurbiprofen to one eye and vehicle solution to the contralateral control eye, while the other 14 received 1% prednisolone acetate to one eye and vehicle solution to the contralateral control eye four times each day in a double-blind fashion. The eyes were photographed and graded weekly with a standardized slit-lamp grading method. After 6 weeks, inhibition of corneal neovascularization was found with flurbiprofen (p = 0.001), while there was a trend toward inhibition of corneal neovascularization with prednisolone acetate (p = 0.076). We also found a significant vehicle effect, with hydroxypropyl methylcellulose, the more viscous vehicle, suppressing corneal neovascularization more than polyvinyl alcohol (p = 0.004).

Platinum spatula versus Mini-tip Culturette in culturing bacterial keratitis.

PURPOSE: To compare the traditional method of culturing bacterial keratitis (platinum spatula) with the use of a commercially available Mini-tip Culturette (Becton-Dickinson, Cockeysville, MD, U.S.A.). METHODS: An experimental model of bacterial keratitis was created in rabbit corneas by intrastromal injection of bacteria. Cultures were taken of rabbit corneas with both the Mini-tip Culturette and the platinum spatula. Culture results were compared with corneal colony counts. Humans with community-acquired presumed bacterial keratitis were cultured with both the Mini-tip Culturette and the platinum spatula. The sensitivity and specificity of the Mini-tip Culturette method was determined and compared with the platinum-spatula technique. RESULTS: Rabbit keratitis model: 100% of corneas had established infections by colony count. Each ulcer was culture positive with platinum spatula, moist Mini-tip Culturette, and dry Mini-tip Culturette. Human keratitis: Seven patients had culture-negative keratitis with both the Mini-tip Culturette and the platinum spatula. Five patients were culture positive with both the Mini-tip Culturette and the platinum spatula. One of the positive cultures had growth of multiple organisms by using the platinum spatula but not with the Mini-tip Culturette. The sensitivity of the Mini-tip Culturette was 83.3%. The specificity of the Mini-tip Culturette was 100%. Detected organisms included group A beta-hemolytic Streptococcus, S. aureus, coagulase-negative Staphylococcus, Serratia marcescens, and Pseudomonas aeruginosa. CONCLUSION: The Mini-tip Culturette is a highly specific and moderately sensitive method for culturing bacterial keratitis.

The role of cultures in the management of ulcerative keratitis.

PURPOSE: To ascertain the importance of routine cultures and gram stains in the management of ulcerative keratitis. METHODS: We retrospectively reviewed 119 consecutive corneal ulcers seen at Sinai Hospital of Baltimore. Cultures were obtained of the corneal ulcer and of the lids and conjunctivae of both eyes. Gram stains were performed by the hospital microbiology department on corneal scrapings from each ulcer. RESULTS: Positive corneal cultures were obtained from 56 eyes (47.1%). Initial antibiotic therapy was changed based on culture results in 14.3% of culture-positive eyes that demonstrated a worsening clinical course. Gram stains were negative in all cases. The sensitivity and specificity of the lid and conjunctival cultures were determined. CONCLUSIONS: Corneal cultures are important in the management of ulcerative keratitis. Lid and conjunctival cultures have low sensitivity and specificity.

A comparison of high-dose continuous and split-course irradiation in non-oat-cell carcinoma of the lung.

From 1969 through 1979, 171 patients with localized but inoperable or unresectable non-oat-cell carcinoma (NOCC) of the lung completed high-dose definitive irradiation. One hundred fifteen received continuous course irradiation to 6000-6500 rad at 180-200 rad/day. Fifty-six received split-course irradiation to 5500 rad at 250-300 rad/day, which included a 2-week break. The two groups were similar with respect to all measured variables. There were no differences in the response rates, failure patterns, survival, or complication rates between the two regimens. The 5-year survival was 6%, with 25.8% dying with infield failure alone and 54.7% with metastases. The incidence of complications was 8.2%, predominantly acute radiation pneumonitis. A review of the most comparable literature reveals no significant improvement in the cure rate of definitively irradiated NOCC with increasing tumor dose, split-course irradiation, or other modifications of radiotherapeutic technique over the past 25 years. The problems of frequent local recurrences and distant metastases, and the poor response of NOCC to presently available systemic therapy, requires that more effective and broadly applicable local and systemic therapies be developed before substantial improvements in the cure rate of NOCC can be expected.