Genomic organization of the region spanning D14Mit262 and D14Mit86 on mouse chromosome 14 and exclusion of Adam28 and Adamdec1 as the cataract-causing gene, lr2.

Mice with recessive cataract, CXSD, show the first clinical symptoms of cataract at five weeks, with complete penetrance. We previously localized the cataract-causing lens rupture 2 gene (lr2) to mouse chromosome 14. In the process of positional cloning of the lr2 gene, we determined the genomic organization of the critical region, defined by D14Mit262 and D14Mit86, and compared it to recently published map information. In addition, mutational analysis using reverse transcription polymerase chain reaction (RT-PCR) followed by direct sequencing as well as quantitative realtime PCR (RQ-PCR) was performed to investigate Adam28 and Adamdec1 as lr2 candidate genes in this study. There was no mutation cosegregating with the phenotype of CXSD mice, which excluded these genes as the lr2 gene. Identification of more transcripts from this region and their mutation analyses are required to isolate the lr2 gene.

Genetic analysis of resistance to radiation lymphomagenesis with recombinant inbred strains of mice.

Induction of lymphomas by radiation in mice is controlled by genetic factors. We analyzed the genetic control of radiation lymphomagenesis using the CXS series of recombinant inbred strains derived from two progenitor strains: one highly susceptible to radiation induction of lymphoma [BALB/cHeA (C)] and one extremely resistant [STS/A (S)]. The best concordances between strain distribution patterns of genetic markers and resistance (or susceptibility) to radiation lymphomagenesis were observed in a region with the b and Ifa genes on chromosome 4. This indicates that one major locus controls the incidence of radiogenic lymphomas in mice. We designated this locus as the Lyr (lymphoma resistance) locus. Backcrosses of (CXS)F1 to the two progenitor strains showed an intermediate incidence of lymphomas between their parental mice and did not significantly differ from (CXS)F1 mice. This and previous observations that (CXS)F1 mice also showed an intermediate incidence, differing from both progenitor strains, indicate that more genes are involved in the resistance (or susceptibility) to lymphoma induced by irradiation.

Variations in Prkdc encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and susceptibility to radiation-induced apoptosis and lymphomagenesis.

DNA double-strand breaks (DSBs) induced by ionizing radiation enforce cells to die, if unrepaired; while if misrepaired, DSBs may cause malignant transformation. The DSB repair system predominant in mammals requires DNA-dependent protein kinase (DNA-PK). Previously, we identified the apoptosis susceptibility gene Radiation-induced apoptosis 1 (Rapop1) on mouse chromosome 16. The STS/A (STS) allele at Rapop1 leads to decreased sensitivity to apoptosis in the BALB/cHeA (BALB/c) background. In the present study, we established Rapop1 congenic strains C.S-R1 and C.S-R1L, which contain the STS genome in a 0.45 cM interval critical for Rapop1 in common in the BALB/c background. Within the segment critical for Rapop1, Prkdc encoding the catalytic subunit of DNA-PK (DNA-PKcs) was assigned. Two variations T6,418C and G11,530A, which induce amino acid substitutions C2,140R downstream from the putative leucine zipper motif and V3,844M near the kinase domain, respectively, were found between BALB/c and STS for Prkdc. The majority of inbred strains such as C57BL/6J carried the STS allele at Prkdc; a few strains including 129/SvJ and C.B17 carried the BALB/c allele. DNA-PK activity as well as DNA-PKcs expression was profoundly diminished in BALB/c and 129/SvJ mice as compared with C57BL/6 and C.S-R1 mice. In the crosses (C.S-R1 x BALB/c)F(1) x 129/SvJ and (C.S-R1 x BALB/c)F(1) x C.B17, enhanced apoptosis occurred in the absence of the wild-type allele at Prkdc. C.S-R1 and C.S-R1L were both less sensitive to radiation lymphomagenesis than BALB/c. Our study provides strong evidence for Prkdc as a candidate for Rapop1 and a susceptibility gene for radiation lymphomagenesis as well.

Modulations of glucocorticoid-induced apoptosis linked to the p53 deletion and to the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1).

We have analysed the effects of p53 and of the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1) located on chromosome 16 on glucocorticoid- and radiation-induced in vivo apoptosis of thymocytes. For those analyses, we used Rapop1 semicongenic mice heterozygous for the STS and BALB/cHeA alleles in the chromosomal segment containing Rapop1 in the BALB/cHeA background, mice bearing a p53 deficient allele in the BALB/cHeA background and the genetic crosses between these mice. The p53 wild type mice with a STS/A allele at the Rapop1 locus were less susceptible to both radiation- and glucocorticoid-induced apoptosis than those with homozygous BALB/cHeA alleles at this locus. Surprisingly, glucocorticoid-induced apoptosis was enhanced in the p53 hemizygous mice and considerably increased in the p53 nullizygous mice. In contrast, a sizable reduction of radiation-induced apoptosis was seen in the p53 hemizygous mice. The low susceptiblity to glucocortocoid-induced apoptosis linked to the STS allele of Rapop1 was less pronounced in the p53 hemizygous mice and a diminished effect of Rapop1 on radiation-induced apoptosis was seen in these mice. Although it remains to be established whether the genes modulating glucocortocoid-induced apoptosis are identical to p53 and Rapop1, our data suggest that p53 and Rapop1 may participate in glucocorticoid-induced apoptosis of thymocytes.

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 gamma-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(-/+) and 13% for p53(+/+), suggesting co-operative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

DNA rearrangements of the int region in spontaneous mouse mammary tumors of SHN/S and SLN/S mice.

SHN and SLN mice originating from the same Swiss albino stock are genetically very close to each other. The incidence and latent period of mammary tumor development in SHN mice were higher and shorter than those in SLN. To elucidate these differences in the behavior of mammary tumorigenesis, the frequency of insertion of mammary tumor viral genes within the int-1 and int-2 regions in spontaneous mammary tumors from their two substrains, SHN/S and SLN/S, were compared. The frequency of provirus integration into either int-1 or int-2 in DNAs from mammary tumors was 52% (11/21) in SHN/S and 45% (5/11) in SLN/S. The frequency of insertion within int-1 or int-2 could not account for the different susceptibilities of SHN/S and SLN/S.

Herpetic keratitis in zinc-deficient rabbits.

In a study of 57 rabbits, we found that zinc played a vital role in the growth of the rabbit and in its immunocompetence. Both humoral and cellular immunity were depressed in the zinc-deficient rabbit. Not only did zinc-deficient animals fail to gain weight, differing in that respect from rabbits on a regular diet, but they also had a high death rate, in contrast with no deaths among normal rabbits. Herpes simplex virus keratitis, both epithelial and stromal, was more severe in the zinc-deficient rabbit but was not improved by local zinc replacement (zinc sulphate ointment, 0.05%); Zinc sulphate ointment (1%), which is four times stronger than commercially available zinc ophthalmic preparations, was severely toxic for the rabbit cornea.

Recombinant human interferon alpha D in HSV-1 recurrence in the rabbit.

Recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing the shedding of herpes simplex virus type-1 (HSV-1) induced by 6-hydroxydopamine iontophoresis followed by topical epinephrine application in previously infected rabbit corneas. A treatment schedule of RIFN alpha D, two drops QID was superior to one drop BID. RIFN alpha A also appeared to be effective in reducing viral shedding. Rabbits treated with RIFN alpha D during two episodes of adrenergically induced HSV-1 shedding, but not during anticipated episodes of spontaneous shedding, did not show a significant reduction in shedding of virus. Interferon was present in significantly higher concentration in tear samples following treatment with RIFN alpha D as compared with RIFN alpha A.

The effect of K-582, a new antifungal agent, on experimental Candida keratitis.

K-582, a new basic peptide antibiotic, was tested in rabbits with experimental Candida keratitis. It was shown that the K-582-treated group showed statistically highly significant therapeutic effects on days 2 and 3, as compared with the control group (day 2: P less than 0.001; day 3: P less than 0.001). The culture study showed that the average number of colonies was 1,573.1 in the controls and 463.3 in the treated group, and the difference was highly significant statistically (P less than 0.001). No ocular or systemic toxic effects were observed with this drug. K-582 is a promising new drug for the treatment of Candida keratitis.

Frequent loss of heterozygosity on chromosomes 4, 12 and 19 in radiation-induced lymphomas in mice.

We found frequent loss of heterozygosity (LOH) on chromosomes 4, 12 and 19 in radiation-induced lymphomas from (BALB/cHeA x STS/A) F1 hybrid mice by allelotype analysis at polymorphic microsatellite loci. The incidences of LOH were 27% (20 of 74 lymphomas), 57% (42 of 74 lymphomas) and 50% (37 of 74 lymphomas) on chromosomes 4 (at D4Mit31), 12 (at D12Mit17) and 19 (at D19Mit11), respectively. These frequent LOH regions are homologous to human chromosomes 9p and 1p, chromosome 12q32.1 and chromosome 10q, respectively. Strain-specific preferential allele loss was observed only on chromosome 4. However, no bias in the frequency of loss between alleles of maternal and paternal origin was observed, indicating that genomic imprinting may not be predominantly involved in these lymphomas. The results suggest that these three regions might harbor tumor suppressor genes responsible for this lymphomagenesis.

Analysis of highly frequent allelic loss region on distal chromosome 12 in murine radiation-induced lymphomas.

Recent genetic studies of tumorigenesis have strongly suggested an existence of tumor suppressor gene(s) on murine chromosome 12 and human chromosome 14q32. We previously described that putative tumor suppressor gene(s) might reside between D12Mit53 and D12Mit233. We analyzed three genes, Tcl1, Yy1 and Tnfalphaip2, which had been mapped around the region, as the candidates in radiation lymphomagenesis of (BALB/c x MSM/Ms)F1 hybrid mice. The locus order and distances of the three genes and microsatellite loci were estimated as follows: [centromere] - Tcl1-(> or =0.085 cM)-D12Mit50-(0.085 cM)D12Mit132-(1.96 cM)D12Mit122-(0.085 cM)D12Mit53-(1.37 cM)-[D12Mit233,D12Mit279,Yy1]-(0.085 cM)-D12Mit181-(> or =0.17 cM)-Tnfalphaip2 - [telomere]. Allele losses at Tcl1, Yy1 genes and D12Mit233 were observed in 94(45%), 143(68%) and 147(70%) of 210 lymphomas, respectively. In semi-quantitative analysis of Yy1 mRNA levels by RT-PCR, kinetics of the yield of the Yy1-cDNA-specific PCR products showed almost the same profiles among thymic lymphomas with allelic loss at Yy1, lymphomas with both alleles retained and normal thymus. These results suggest that Tcl1, Yy1 and Tnfalphaip2 genes are not predominantly involved in radiation lymphomagenesis of mice. In further analysis of the common allelic loss region, we found new loci, Y152pR1 and Y184pR2, from YACs which located in the hot region between D12Mit53 and D12Mit233, and the highest frequency of allelic loss (71%) was observed at the Y184pR2 locus. The LOH patterns of individual lymphomas suggest that putative tumor suppressor gene(s) lies between Y152pR1 and Y184pR2.

Hormone-dependent human tumors express the fms gene-product.

Human mammary and thyroid tumors as well as peripheral blood cells of the same patients were examined for the amplification of oncogene fins and the expression of its product. Of the 7 mammary tumors analyzed, amplification of fms was observed in 6 (86%) mammary tumor DNAs, while no amplification was seen in 5 thyroid tumors and 7 peripheral blood cell DNAs tested. None of the mammary and thyroid tumors showed any rearrangement of the fms gene. Investigations of the expression of the product of fms in various human tumors were carried out following the method of immunohistochemical staining in which polyclonal antibody to the fms gene product was used. The incidence of the expression of fms gene product in the tumors of mammary and thyroid glands, prostate and ovary, the hormone-dependent organs, was 38-97%, whereas fms protein was found to be present only in 20 and 42% of the tumors from hormone-independent organs, the brain and the bladder. Expression of the fms gene product was not detectable in normal tissue surrounding the tumor tissue in any of the cases examined. These results suggest that the expression of the product of fms may be associated with the development of some tumors of hormone responsive organs, especially the breast and thus the fms gene product may be a valuable marker for human mammary tumors.

Staphylococcal blepharitis.

A detailed discussion of various aspects of staphylococcal blepharitis is presented. These include epidemiology, pathogenesis and immunity, microbiologic characteristics, clinical signs, associated systemic diseases, differential diagnosis, and treatment.

Treatment of herpes simplex keratitis with levamisole.

A model of chronic herpetic keratitis was developed by injecting rabbits subconjunctivally with a corticosteroid. A levamisole hydrochloride-treated group of these rabbits developed milder, more rapidly healing epithelial lesions than an untreated group. Although most of the untreated rabbits developed stromal disease, almost all of the lesions in the levamisole-treated animals were limited to the epithelium.

Tretinoin and corneal epithelial wound healing.

The entire corneal epithelium of each of 16 rabbits was removed bilaterally. Tretinoin (vitamin A acid) ointment was applied topically twice daily to the eyes of one half of the experimental animals, and the ointment base alone was applied to the eyes of the other half (the controls). On days 1, 2, and 3 after the removal of the epithelium, the healing of the denuded corneas of the animals receiving tretinoin was significantly (P = .01, .01, and .05, respectively) more advanced than the healing of the corneas of the control animals.

Combined amphotericin B and rifampin treatment of experimental Candida albicans keratitis.

In a model of experimental Candida albicans keratitis in rabbits, treatment with a combination of amphotericin B and rifampin was compared with treatment with amphotericin B alone. Both modes of therapy substantially reduced the number of organisms in the cornea below the number in untreated control corneas. In the group treated with combined therapy, there were significantly fewer organisms in the cornea after three days of therapy than in the group treated with amphotericin B alone. The results of this study indicate that the treatment of C albicans keratitis in rabbits with combined amphotericin B and rifampin is more effective than treatment with amphotericin B alone.

Diagnosis of allergic conjunctivitis.

Itching was cited as a major symptom of their ocular disease by 49 (80%) of the 61 patients with allergic conjunctivitis. Conjunctival scrapings from 51 patients (84%) demonstrated intact eosinophils or eosinophil granules. Eosinophil granules were a useful and recognizable cytologic feature of allergic conjunctivitis even in the absence of intact eosinophils. We believe that a history of itching and the presence of eosinophils or eosinophil granules in conjunctival scrapings are helpful in diagnosing allergic conjunctivitis.

Combined gentamicin-tobramycin-corticosteroid treatment. II. Effect on gentamicin-resistant Pseudomonas keratitis.

In an experimental model of Pseudomonas keratitis produced by a gentamicin sulfate-resistant, tobramycin sulfate-sensitive strain of P aeruginosa, the results of two treatment regimens-(1) with gentamicin and tobramycin alone and (2) with gentamicin, tobramycin, and a steroid-were evaluated. All of the animals exhibited ultimately the same amount of corneal inflammation and corneal scarring. In the group receiving the antibiotic-steroid combination, the corneal lesions healed more slowly.

Herpes simplex keratitis treatment with vitamin A.

We produced chronic experimental herpetic keratitis by dropping PH-strain herpes simplex virus on scarified rabbit corneas and then injecting the rabbits subconjunctivally with low doses of corticosteroid (namely, triamcinolone acetonide suspension). Vitamin-A-treated rabbits developed milder, more rapidly healing epithelial lesions than untreated rabbits. Whereas most of the untreated rabbits developed moderate or severe stromal disease, most of the vitamin-A-treated rabbits developed only mild stromal disease or none at all.

Quantitative antibiotic sensitivity determinations of Staphylococcus aureus isolated from eye cultures.

The sensitivities of 108 strains of Staphylococcus aureus, isolated from patients with a variety of ocular conditions, were tested quantitatively against nine antibiotics by means of a microtiter broth-dilution method. Of the nine antibiotics, cephalothin sodium had the lowest (and therefore the best) minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC). The MBC and MIC of gentamicin sulfate were very close to those of cephalothin. The MBCs and MICs of erythromycin, ampicillin sodium, penicillin, tetracycline, methicillin sodium, and carbenicillin disodium were moderately good. Chloramphenicol had the poorest response.