Transforming growth factor-ß1 sustains the survival of Foxp3(+) regulatory cells during late phase of oropharyngeal candidiasis infection.

As CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play crucial immunomodulatory roles during infections, one key question is how these cells are controlled during antimicrobial immune responses. Mechanisms controlling their homeostasis are central to ensure efficient protection against pathogens, as well as to control infection-associated immunopathology. Here we studied how their viability is regulated in the context of mouse oropharyngeal candidiasis (OPC) infection, and found that these cells show increased protection from apoptosis during late phase of infection and reinfection. Tregs underwent reduced cell death because they are refractory to T cell receptor restimulation-induced cell death (RICD). We confirmed their resistance to RICD, using mouse and human Tregs in vitro, and by inducing α-CD3 antibody-mediated apoptosis in vivo. The enhanced viability is dependent on increased transforming growth factor-ß1 (TGF-ß1) signaling that results in upregulation of cFLIP (cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein) in Tregs. Protection from cell death is abrogated in the absence of TGF-ß1 signaling in Tregs during OPC infection. Taken together, our data unravel the previously unrecognized role of TGF-ß1 in promoting Treg viability, coinciding with the pronounced immunomodulatory role of these cells during later phase of OPC infection, and possibly other mucosal infections.

The key role of aquaporin 3 and aquaporin 10 in the pathogenesis of pompholyx.

Pompholyx remains a chronic skin affliction without a compelling pathophysiological explanation. The disease is characterized by the sudden onset of vesicles exclusively in the palms and soles which generally resolves. However, the disease may progress and the vesicles may expand and fuse; with chronicity there is deep fissuring. Multiple therapeutic approaches are available, but the disease is often resistant to conventional treatments. Currently, oral alitretinoin is used for patients with resistant chronic disease; however, this therapy is only approved for use in the UK, Europe and Canada. In this paper we wish to put forward a hypothesis: exposure to water and the subsequent steep osmotic gradient imbalance are key factors driving skin dehydration seen in pompholyx patients once the disease becomes chronic. The mechanistic explanation for the epidermal fissuring might lie in the over-expression across the mid and upper epidermis, including the stratum corneum, of two water/glycerol channel proteins aquaporin 3 and aquaporin 10, expressed in the keratinocytes of afflicted pompholyx patients. The over-expression of these two aquaporins may bridge the abundantly hydrated dermis and basal epidermis to the outer environment allowing cutaneous water and glycerol to flow outward. The beneficial effects reported in alitretinoin-treated patients with chronic hand eczemas may be due potential regulation of aquaporin 3 and aquaporin 10 by alitretinoin.

Novel cell type-specific antiviral mechanism of interferon gamma action in macrophages.

Interferon (IFN)-gamma and macrophages (Mphi) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-gamma mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mphi (BMMphi). IFN-gamma inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1alpha-dependent manner much more effectively in BMMphi (approximately 100-fold) than MEF (5-10-fold). Although initial STAT-1alpha activation by IFN-gamma was equivalent in MEF and BMMphi, microarray analysis demonstrated that IFN-gamma regulates different sets of genes in BMMphi compared with MEFs. IFN-gamma inhibition of MCMV growth was independent of known mechanisms involving IFN-alpha/beta, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-gamma-induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-gamma action, which differed in MEF and BMMphi. In BMMphi, IFN-gamma reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-gamma on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-gamma had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-gamma action restricted to Mphi, a cell type key for MCMV pathogenesis and latency.

The Cpx envelope stress response is controlled by amplification and feedback inhibition.

In Escherichia coli, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the cpxRA operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription of cpxRA. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.

Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres.

Gene silencing is required to stably maintain distinct patterns of gene expression during eukaryotic development and has been correlated with the induction of chromatin domains that restrict gene activity. We describe the isolation of human (EZH2) and mouse (Ezh1) homologues of the Drosophila Polycomb-group (Pc-G) gene Enhancer of zeste [E(z)], a crucial regulator of homeotic gene expression implicated in the assembly of repressive protein complexes in chromatin. Mammalian homologues of E(z) are encoded by two distinct loci in mouse and man, and the two murine Ezh genes display complementary expression profiles during mouse development. The E(z) gene family reveals a striking functional conservation in mediating gene repression in eukaryotic chromatin: extra gene copies of human EZH2 or Drosophila E(z) in transgenic flies enhance position effect variegation of the heterochromatin-associated white gene, and expression of either human EZH2 or murine Ezh1 restores gene repression in Saccharomyces cerevisiae mutants that are impaired in telomeric silencing. Together, these data provide a functional link between Pc-G-dependent gene repression and inactive chromatin domains, and indicate that silencing mechanism(s) may be broadly conserved in eukaryotes.

Isolation and characterization of an isogenic set of Salmonella typhimurium strains analogous to the "Ames" tester strains.

The standard Ames tester strains of Salmonella typhimurium contain a number of genetic differences at loci other than his. The fact that these strains contain independently isolated uvrB-bio-gal deletions and rfa mutations implies that these are likely to vary from strain to strain. Since the strains were isolated from different parental stocks of S. typhimurium LT-2, they differ in their ability to metabolize arabinose. Other, unknown differences may exist because the isolation of some of the strains involved ultraviolet and chemical mutagenesis. We have isolated a set of isogenic S. typhimurium strains that contain the relevant genetic markers of the standard Ames tester strains. These strains all contain the same uvrB-bio-gal deletion and the same rfa mutation; they differ only in the nature of their his mutations and in the presence or absence of the plasmid pKM101. We have assessed the responsiveness of these strains to a number of mutagens and conclude that their mutagenic specificities are the same as those of the corresponding Ames strains: TA98, TA100, TA1535, TA1537 and TA1538. Therefore, the specificity of the standard Ames strains with respect to these mutagens is a result solely of the differences in the nature of their his mutations and the effects of pKM101.

Colposcopy.

Colposcopy was introduced to Canadian medical practice in the late 1960s and its use has only recently become widespread. Until the advent of colposcopy, most women with abnormal Pap smears had a cone biopsy of the cervix with all its attendant problems. The colposcope has completely changed the management of an abnormal Pap smear with respect to diagnosis and has had a remarkable influence on treatment. Cryosurgery and carbon dioxide laser surgery were introduced as out-patient treatments for precursors of cervical cancer because by using colposcopy the physician is able to accurately diagnose cervical intraepithelial neoplasia. Colposcopy has also been an important tool in the detection, diagnosis, and treatment of human papillomavirus infections in men and women.

Effects of pH on weak and positive control mutagens in the Ames Salmonella plate assay.

The effects of pH on the mutagenic activity of several chemicals were evaluated in the standard Ames Salmonella typhimurium plate-incorporation assay. The pH of the base agar was varied between 6.0 and 8.0. The positive control compounds routinely used in this laboratory, 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and nitrofurantoin, showed increasing mutagenic activity as the pH was decreased to 6.0. However, the activity of two weakly mutagenic cosmetic ingredients, 2,2',4,4'-tetrahydroxybenzophenone and trans-4-phenyl-3-buten-2-one, was completely eliminated at pH levels near 6.0. It is concluded that plates poured with agar with pH levels below 7.0 can result in strong responses for the positive control chemicals but give negative results for some mutagens.

The changing concepts of condyloma. A retrospective study of colposcopically directed cervical biopsies.

Human papillomavirus has been implicated in the etiology of cervical intraepithelial neoplasia. A retrospective audit was conducted on all colposcopically directed biopsies performed from June through November, inclusive, in the years 1980 and 1982, a total of 317 cases. In 1980, the misdiagnosis rate for condyloma was 88.9% (69 of 78) and, in 1982, the misdiagnosis rate was reduced to 28.5% (40 of 140). On review, the association of cervical intraepithelial neoplasia with condyloma was 73% (107 of 146), p less than 0.005. The differences in mean ages of patients with condyloma alone, cervical intraepithelial neoplasia with condyloma, and cervical intraepithelial neoplasia alone were significant, p less than 0.02. When condyloma is not recognized, the pathologist tends to overdiagnose grade 3 cervical intraepithelial neoplasia as shown in 28 original cases; 16 of 28 (57.1%) were condyloma alone on review. Fifteen (53.6%) of these 28 patients underwent a cone biopsy or hysterectomy when a less radical procedure would have been acceptable. The morphologic changes indicating cervical condyloma were not fully appreciated by the pathologist, and this resulted in overdiagnosis and later overtreatment by the gynecologist.

Infertility surgery with the laser beam.

The results of the use of CO2 laser in infertility surgery are encouraging. The unique properties of CO2 laser--precision of application, minimal bleeding, rapid healing, and minimal scarring--are beneficial for infertility surgery. It remains to be seen, however, whether the pregnancy rate in "laser patients" is superior to that in "conventional surgical patients." A well-controlled, randomized study and a long-term follow-up period are required to clarify this matter. A good understanding of the basic concepts of laser physics and a familiarity with the laser instruments are mandatory for the laser surgeon. A well-informed operating room staff will certainly be helpful in conducting effective and safe laser surgery. For example, all flammable and explosive material must be removed from the laser area, and protective coverings for the eyes must be worn by all personnel. Simplification, reduction in the bulk of the present instruments, and the development of a flexible CO2 laser transmission fiber that can be utilized through an endoscope will be beneficial.

Treatment of cervical intraepithelial neoplasia with the carbon dioxide laser.

The carbon dioxide laser was used to treat 138 patients with cervical intraepithelial neoplasia between February, 1979, and February, 1981. All patients in this study were assessed in the colposcopy clinic prior to treatment. The diagnosis of cervical intraepithelial neoplasia was based on colposcopic examination, cytologic findings, colposcopically directed biopsy, and endocervical biopsy in all cases. Included in the 138 patients were 26 with grade 1, 82 with grade 2, and 30 with grade 3 cervical intraepithelial neoplasia. The persistence rate of abnormal cytologic findings at 7 months after treatment was 11% for grade 1, 8.5% for grade 2, and 10% for grade 3 cervical intraepithelial neoplasia. No major complications accompany this treatment modality, which was performed in the outpatient clinic without the use of anesthesia or analgesia.

Metronidazole in the prophylaxis of anaerobic infections in gynecologic surgery.

A double-blind study was initiated in January 1978 to determine the effectiveness of the prophylactic use of metronidazole, either orally or intravenously, in patients undergoing elective gynecologic surgery. A total of 121 women were evaluated; 42 women received oral metronidazole, 40 women intravenous metronidazole, and 39 women placebo therapy. Evidence of postoperative infection was present in 20% of patients treated with placebo, whereas in the active drug groups the incidence of infection was 12%. Blood levels of metronidazole during the operations had a mean value of 22.2 and 17.9 micrograms/ml for patients receiving intravenous and oral metronidazole, respectively. Preoperative cultures for anaerobic organisms revealed an incidence range of 23% to 45% for the three study groups. The incidence of positive anaerobic cultures declined dramatically by the second postoperative day for patients receiving intravenous and oral metronidazole, In the placebo group, the incidence was not significantly different from that in the preoperative period. This preliminary study revealed that the incidence of postoperative infections subsequent to gynecologic surgery can be reduced significantly through the use of prophylactic metronidazole.

Evaluation, using Salmonella typhimurium, of the mutagenicity of seven chemicals found in cosmetics.

Six chemicals used as ingredients in cosmetics were evaluated for mutagenic activity in Salmonella typhimurium. Two of these ingredients, trans-4-phenyl-3-butene-2-one and 2,2',4,4'-tetrahydroxybenzophenone, were mutagenic in the presence of rat liver S-9 towards strains TA100 and TA1537 respectively. An impurity found in some cosmetic products, N-nitrosodiethanolamine, was mutagenic to S. typhimurium strains TA1535 and TA100 in the presence of hamster-liver S-9 but not rat-liver S-9.

Diffuse peritoneal seeding in immunoblastic lymphosarcoma.

The authors report a case of immunoblastic lymphosarcoma of B cells with an unusual presentation. Laparotomy on a 67-year-old women with an abdominal mass revealed extensive peritoneal seeding resembling carcinomatosis. At autopsy, immunoblastic lymphosarcoma, probably originating in the bowel wall, was found. It was studied by light, immunofluorescent and electron microscopy. Histologic examination showed monomorphic proliferation of malignant immunoblasts with plasmacytoid features. Immunofluorescent microscopy was positive for intracytoplasmic IgG, IgA, kappa and lambda light chains. The presence of diffuse peritoneal seeding in patients with malignant lymphomas is not well documented. The clinical and pathologic features of this entity and its relation to Mediterranean lymphoma are discussed.

Prolonged amenorrhea and oral contraceptives.

Of 106 consecutive women referred for secondary amenorrhea of more than 1 year's duration, 65 were diagnosed as having functional amenorrhea. Of these 65, 29 had amenorrhea directly following discontinuation of oral contraceptives (OC group) and 36 had never used oral contraceptives (NOC group). There was no difference in the incidence of prior menstrual irregularity in either group. Similarly, there was no difference in the resting serum estrone, estradiol, luteinizing hormone, follicle-stimulating hormone, and prolactin levels between the OC and NOC groups. Nor was there a difference between the OC and NOC groups in response to medroxyprogesterone acetate, clomiphene citrate, or luteinizing hormone-releasing factor. Of 106 patients, 17 were proven to have prolactinomas. Eight patients had a prior history of OC use, whereas nine did not. With the exception of elevated serum prolactin levels, there were no significant differences in biochemical tests or history of oral contraceptive use between the prolactinoma group and patients with prolonged "functional" amenorrhea (OC plus NOC groups). The lack of historical or biochemical difference between the OC and NOC subjects indicates homogeneity between groups, and does not support the existence of a "postpill" syndrome.

PIP: 65 of 106 consecutive women referred for secondary amenorrhea of more than 1 year's duration were diagnosed as having functional amenorrhea. 29 of the 65 women had amenorrhea directly following discontinuation of oral contraceptives (OC group) and 36 had never used OCs (NOC group). There was no difference in the incidence of prior menstrual irregularity in either group, and there was no difference in the resting serum estrone, estradiol, luteinizing hormone, follicle-stimulating hormone, and prolactin levels between the 2 groups. There was no difference between the groups in response to medroxyprogesterone acetate, clomiphene citrate, or luteinizing hormone-releasing factor. 17 of the 106 patients were proven to have prolactinomas; 8 of these had a prior history of OC use and 9 did not. Except for elevated serum prolactin levels, there was no significant differences in biochemical tests or history of OC use between the prolactinoma group and patients with prolonged "functional" amenorrhea (OC plus NOC groups). The lack of historical or biochemical difference between the OC and NOC subjects suggests homogeneity between groups and fails to support the existence of a "postpill" syndrome.

Early diagnosis of ovarian cancer.

Ovarian cancer is the most common fatal gynecologic malignant disease. Unfortunately 60% to 70% of patients present initially with advanced disease. Progress in the treatment of this condition will be made only if it can be diagnosed early. Pelvic examination is still the best screening test despite attempts to make an early diagnosis with the use of cytology, ultrasonography, and serum determinations for fibrin degradation product; haptoglobin, protein-bound fucose and tumour-associated antigens. The best prospect for a screening test for ovarian cancer probably lies in the field of tumour immunology.