1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes.

The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3. Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.

Antiestrogen potentiation of antiproliferative effects of vitamin D3 analogues in breast cancer cells.

[3H]thymidine incorporation and DNA content were used to investigate the antiproliferative effects of 1,25(OH)2D3 and four analogues [16-ene-1,25(OH)2D3 (16-ene)]; 16-ene,23-yne-1,25(0H)2,D3; EB1089; and 22 oxa-1,25(OH)2D3] on MCF-7, BT-474, and MDA-MB-453 breast cancer cell lines. 1,25(OH)2D3 and the analogues elicited a biphasic response from MCF-7 and BT-474 estrogen receptor (ER)-positive cells, in the presence of estradiol (E2), with lower doses (between 10(-12) and 10(-10) M) tending to stimulate proliferation and higher doses (between 10(-9) and 10(-6) M) inhibiting proliferation by as much as 65%. In the absence of E2, the stimulatory effect was abrogated. Proliferation of MDA-MB-453, estrogen receptor-negative (ER-) cells, was stimulated by these compounds only at 10(-12) M, and inhibited by all higher doses, by as much as 83%. All three cell lines were shown to be vitamin D receptor (VDR) positive, and 1,25(OH)2D3 and all four analogues bound to the VDR with high affinities in each cell line. The antiestrogen ICI 164,384 inhibited the proliferation of all three cell lines. ICI 164,384 at 10(-8) M in combination with 1,25(OH)2D, or EB1089 converted biphasic response of the ER+ cells to one resembling the response of the ER- cells, by eliminating the stimulatory response elicited by 1,25(OH)2D3 at low doses and enhancing the antiproliferative effects of higher doses by as much as 1000-fold. These data are consistent with the hypothesis that E2 in the ER+ cells blocks the antiproliferative effects of the analogues and suggest the potential usefulness of combined antiestrogen and 1,25(OH)2D3 analogues in ER+ breast tumors, whereas 1,25(OH)2D3 analogues alone might suffice in ER- breast tumors.

Squamous carcinoma cell lines fail to respond to 1,25-Dihydroxyvitamin D despite normal levels of the vitamin D receptor.

Squamous carcinoma cells (SCC) fail to differentiate under conditions that are favorable for the growth and differentiation of normal human keratinocytes. Human keratinocytes differentiate from a highly proliferative basal cell to a terminally differentiated cornified cell in culture in the presence of physiological levels of extracellular calcium. 1,25-Dihydroxyvitamin D (1,25[OH]2D3) potentiates this process. Previous studies have shown that the differentiation process in keratinocytes is associated with increased expression of the genes for involucrin and transglutaminase, the products of which participate in cornified envelope formation. The mRNA for involucrin and transglutaminase was not detected in the SCC lines studied (viz. SCC4, 12B2, 12F2, A431, and HACAT) when they were grown in serum free medium. Addition of at least 2% fetal bovine serum for 48 h triggered the expression of these genes, which could then be maintained in the absence of serum. Serum was not required for induction of these genes in keratinocytes. In these cells, 1,25(OH)2D3 stimulated the expression of involucrin and transglutaminase in a concentration-dependent manner, while the SCC lines failed to respond to 1,25(OH)2D3 regardless of whether these cells had been pre-exposed to serum. An important factor that mediates 1,25(OH)2D3-stimulated gene expression is the vitamin D receptor, but vitamin D receptor mRNA levels in all the SCC lines examined were comparable to those in keratinocytes. Furthermore, the vitamin D receptor protein levels in SCC lines as assessed by ligand-binding analysis were comparable to those of keratinocytes. Thus, the mediators of 1,25(OH)2D3 action on gene expression other than the vitamin D receptor may be missing or defective in SCC lines, whereas the mediators of as yet undefined agents in serum may be better expressed in SCC lines than in keratinocytes. Our results indicate that, although SCC lines are capable of expressing the genes for the proteins involved in differentiation, the control of the expression of these genes by 1,25(OH)2D3 is abnormal in SCC despite the presence of a functional vitamin D receptor in concentrations equivalent to those in keratinocytes.

Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor.

Extracellular calcium concentrations (Cao) > 0.1 mM are required for the differentiation of normal human keratinocytes in culture. Increments in Cao result in acute and sustained increases in the intracellular calcium level (Cai), postulated to involve both a release of calcium from intracellular stores and a subsequent increase in calcium influx through nonspecific cation channels. The sustained rise in Cai appears to be necessary for keratinocyte differentiation. To understand the mechanism by which keratinocytes respond to Cao, we measured the acute effects of Cao on Cai and calcium influx in keratinocytes at various stages of differentiation. We then demonstrated the existence of the calcium receptor (CaR) in keratinocytes and determined the effect of calcium-induced differentiation on its mRNA levels. Finally, we examined the role of Cai in regulating both the initial rise in Cai after the switch to higher Cao and the activity of the nonspecific cation channel through which calcium influx occurs. Our data indicate that the acute Cai response to Cao is lost as the cells differentiate and increase their basal Cai. These data correlated with the decrease in CaR mRNA levels in cells grown in low calcium. However, calcium influx as measured by 45Ca uptake increased with differentiation in 1.2mM calcium, consistent with the increase in CaR mRNA in these cells as well as the calcium-induced opening of the nonspecific cation channels. We conclude that the keratinocyte contains a CaR that regulates both the initial release of Cai from intracellular stores and the subsequent increase in calcium flux through nonspecific calcium channels. A rising level of Cai may turn off the release of calcium from intracellular stores while potentiating the influx through the nonspecific cation channels. Differentiation of keratinocytes appears to increase the CaR, which may facilitate the maintenance of the high Cai required for differentiation.

Evidence for separate control mechanisms at the message, protein, and enzyme activation levels for transglutaminase during calcium-induced differentiation of normal and transformed human keratinocytes.

We analyzed the effects of three different calcium concentrations on the RNA and functional protein levels of transglutaminase (TGase) and involucrin (INV) over time in culture. We compared the results in normal human keratinocytes with those in a squamous cell carcinoma, SCC4. The highest calcium concentration (1.2 mM) induced the greatest levels of INV and TGase message, INV protein, and rates of CE formation, but not maximal levels of TGase protein. By examining cytosol and membrane fractions of keratinocytes, we found that after synthesis, TGase protein shifts, under the influence of calcium (both 0.1 mM and 1.2 mM), from the cytosol into the membrane in postconfluent cells. However, only 1.2 mM calcium induced significant amounts of TGase activity. These data indicate that elevated calcium (1.2 mM) achieves the expected induction in keratinocyte differentiation by regulation of not only INV and TGase message levels, but also the translation and activation of TGase protein. Our data suggest that this calcium-induced activation of TGase protein occurs while the protein is anchored in the membrane. In contrast, despite ample INV and TGase message levels within SCC4 cells, these RNA levels are not regulated by calcium or translated into protein, suggesting that the transformed phenotype of SCC4 cells results not only in a failure of calcium to regulate gene transcription, but also in a defect within the translation machinery of these differentiation-specific proteins.

1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces the differentiation of normal human keratinocytes, in part by increasing their basal intracellular calcium levels (Cai) over a period of hours. Agonists such as ATP acting through membrane receptors cause an immediate but transient increase in Cai accompanied by an increase in inositol trisphosphate (IP3). Treatment of keratinocytes for 24 h with 1 nM 1,25(OH)2D3 resulted in a two- to four-fold potentiation of the Cai response of these cells to ATP. This potentiation was inhibitable with cycloheximide, unaccompanied by a change in total intracellular calcium pools, but associated with an increase in basal IP3 levels and ATP-stimulated IP3 production. Treatment with 1,25(OH)2D3 raised the protein and mRNA levels of phospholipase C isoenzymes, particularly phospholipase C-beta 1 in a dose-dependent manner. These studies indicate that 1,25(OH)2D3 modulates the keratinocyte signal transduction pathway by induction of phospholipase isoenzymes, a previously undescribed action for this hormone.

Enzyme-linked immunosorbent assays for detection of immunoglobulin M to nontreponemal and treponemal antigens for the diagnosis of congenital syphilis.

Sera from newborn infants born of mothers with a high risk of syphilis were examined for immunoglobulin M (IgM) antibodies in two different enzyme-linked immunosorbent assays (ELISAs), using either purified flagella from Treponema phagedenis biotype Reiter or the Venereal Disease Research Laboratory (VDRL) antigen as the antigen. All sera were also examined by the fluorescent treponemal antibody absorption (FTA-ABS) test for IgM. Three different groups of patients were studied. Group 1 consisted of 84 women and their newborn infants from a high-risk population for syphilis. Congenital syphilis was diagnosed in one child who had an IgM-positive cord blood specimen in both the ELISA and the FTA-ABS test. Group 2 consisted of 10 mothers and their newborn children. All mothers had positive syphilis-screening tests, and all children had signs of congenital syphilis. All but one child had positive IgM tests. Group 3 consisted of 15 mothers and their newborn children. These mothers had been treated for syphilis late in pregnancy, and all had a positive screening test at delivery. Two of the children had positive IgM tests, probably caused by reactivity after late intrauterine treatment of congenital syphilis. The specificities of the IgM tests were high when evaluated with sera from newborn children without signs of congenital syphilis. Even though IgM rheumatoid factor was found in all of the children tested with definite congenital syphilis, the rheumatoid factor did not cause false-positive results in either the VDRL ELISA or the flagellum ELISA. No significant IgG-IgM competition was noticed in the ELISAs. This study also confirmed that IgA antibodies do not cross the placenta; most newborn children with congenital syphilis were positive in the VDRL ELISA for IgA. Both the VDRL ELISA and the flagellum ELISA are very useful in the diagnosis of congenital syphilis and may be substitute for the FTA-ABS test. The VDRL ELISA for IgM will be especially useful in developing countries with a high incidence of congenital syphilis.

Evaluation of a DNA-hybridization method for detection of African and Asian strains of Neisseria gonorrhoeae in men with urethritis.

The 2.6-megadalton (MDa) cryptic plasmid and the 4.4-MDa beta-lactamase plasmid of Neisseria gonorrhoeae were radiolabeled with [32P] nucleotides and used as probes for direct detection of gonococci and beta-lactamase plasmids in urethral exudates from men with urethritis. The sensitivity and specificity of the DNA probes were compared with culture isolation of N. gonorrhoeae and biochemical tests of gonococcal isolates for beta-lactamase production. Of 216 urethral specimens, 180 were positive for N. gonorrhoeae by DNA probe and culture, 27 were negative by both tests, and 9 gave discordant results. Compared with culture and with the chromogenic cephalosporin assay, the sensitivity and the specificity of the DNA probe was 99% and 93% and that of the beta-lactamase probe assay was 91% and 96%, respectively. Electrophoresis of plasmids isolated from 90 gonococcal cultures showed that all contained the 2.6-MDa plasmid, 29 possessed a 3.2-MDa plasmid, 18 a 4.4-MDa beta-lactamase plasmid, and 11 had a 24.5-MDa conjugal plasmid. We conclude that the sensitivity of our DNA probes was comparable to that of culture for diagnosis of gonorrhea and to conventional tests for detection of beta-lactamase.

Syphilis in pregnant women in Zambia.

Because of the high incidence of congenital syphilis at the University Teaching Hospital, Lusaka, Zambia, the potential risks of congenital infection and fetal loss due to syphilis were assessed by screening 202 antenatal patients, 340 pregnant women admitted to the hospital whose pregnancies ended in either spontaneous abortion or stillbirth, and 469 consecutive babies delivered at the hospital. Primary serological screening was performed with the rapid plasma reagin test, and reactive sera were confirmed by the Treponema pallidum haemagglutination test. In all cases detailed histories were obtained and patients were examined for clinical signs of syphilis. The TPHA test result was reactive in 12.5% of antenatal patients and in 42% of women who aborted in the later half of pregnancy. Among 469 consecutive babies delivered at the hospital, 30 had reactive results to the TPHA test; of these two were stillborn and four had signs of congenital syphilis at birth. Thus, syphilis appears to affect adversely an appreciably high number of pregnant women in Zambia. For this reason a special campaign to screen adequately and treat pregnant women and neonates is needed.

PIP: Three groups of patients were investigated: 202 pregnant women who attended a suburban antenatal clinic for the first time; 340 pregnant women whose pregnancies resulted in either spontaneous abortion or stillbirth; and 464 pregnant women admitted to the labor ward of the University Teaching Hospital, Lusaka. The socioeconomic background and relevant obstetric and prenatal histories of each patient were recorded; the patients were examined for clinical signs of syphilis. Blood samples were obtained for preliminary serological screening for treponemal antibodies with the rapid plasma reagin (RPR) test and reactive sera were tested by the Treponema pallidum hemagglutination (TPHA) test. The cerebrospinal fluid was examined routinely for the presence of treponemal antibodies. 85% of the women in the antenatal group were 20 or more weeks pregnant. 173 of the 240 spontaneous abortions occurred before the 20th weeks of pregnancy. Seroreactivity in the TPHA test in these two subgroups was 9.8% and 41.8% respectively, In 36 of the 42 seroreactive women who gave birth to stillborn babies, the presence of syphilis was the only recognizable etiological factor. 35 women had premature deliveries; 40 of the babies were macerated at birth. In the 167 cases of fetal loss beyond the 20th week of pregnancy, there were no significant differences in age, marital and economic status, education, and parity between seropositive and seronegative groups. A history of previous abortion or stillbirth and antenatal reactivity to the Venereal Disease Research Laboratory test was significant in the seroreactive group. Among the 30 seroreactive babies, 4 had clinical signs of congenital syphilis at birth (detected by darkfield microscopy of exudate in 3 babies), 2 were stillborn, and 8 more required intensive care because of prematurity, asphyxia, and conjunctivitis. The overall mortality and morbidity rates in babies whose sera were reactive in the TPHA test were significantly higher (p 0.001) than in those whose sera were not reactive.

Penicillin and spectinomycin in treatment of gonococcal urethritis.

In view of the recent discovery of penicillinase-producing strains of Neisseria gonorrhoeae in Zambia, the efficacies of single intramuscular doses of aqueous procaine penicillin G (4.8 x 10(6) units plus 1 g of oral probenecid) and 2 g of spectinomycin were evaluated in an open clinical trial of the treatment of acute gonococcal urethritis in men. The former regimen was given to 123 men; failure of treatment was observed in 9.1% of the 88 men followed for two weeks. Spectinomycin was given to 124 men; treatment failure occurred in 3.8% of the 104 men followed for two weeks. During the trial, 190 unselected isolates of N. gonorrhoeae were screened by rapid iodometric test, and two penicillinase-producing strains were detected. MICs of penicillin and spectinomycin were determined by the agar dilution method for 110 and 98 isolates, respectively. MICs of penicillin of greater than or equal to 0.125 micrograms/ml were observed with 78.2% of the strains, while 83.7% had MICs of spectinomycin of less than or equal to 15.0 micrograms/ml. It was suggested that penicillin be given routinely for treatment of gonorrhea in Zambia and that spectinomycin be reserved for treatment of gonococcal infections not cured by penicillin.

Penicillinase-producing gonococcal strains in Zambia. Observations on treatment failures.

Penicillinase-producing strains of Neisseria gonorrhoeae (PPNG) were detected in nine out of 27 (3.2%) treatment failures in 310 cases of acute gonococcal urethritis in men in Lusaka, Zambia. Minimum inhibitory concentrations of penicillin for 17.2% of 233 gonococcal isolates were less than or equal to 0.05 microgram/ml, for 38.2% between 0.125 and 0.25 microgram/ml, and for 46.6% greater than or equal to 0.5 microgram/ml. At present the prevalence of PPNG in African countries is not known but is likely to increase rapidly unless simplified control schemes are adopted within the existing health care programmes. Endemic pockets of PPNG in a few countries can threaten worldwide efforts to control gonorrhoea.

Gonococcal infection in women with pelvic inflammatory disease in Lusaka, Zambia.

Gonococcal infection is the most common disease in both male and female patients seen at the Sexually Transmitted Disease Clinic at the University Teaching Hospital in Lusaka, the capital city of Zambia. In this study, 100 women admitted to the gynecology wards for management of pelvic inflammatory disease were screened for gonococcal infection using an endocervical culture on modified Thayer-Martin medium. Forty-six of these women had gonococcal infection, including 65% of those women with PID of less than 2 weeks' duration. Because gonorrhea is so common among women with PID, "standard" antibiotic regimens must be reevaluated. We recommend for countries with a high incidence of gonorrhea that initial treatment of PID should include an antibiotic regimen effective for gonococcal infection.

PIP: The rate of gonococcal infection among PID (pelvic inflammatory disease) patients admitted to the University Teaching Hospital in Lusaka, Zambia was assessed and found to be high. 100 women were selected for study from among the 812 PID patients admitted to the hospital over a 6 month period. Women who were pregnant, who recently delivered or had an abortion, who recently underwent gynecological surgery, who had received antibiotic treatment for their current episode of PID were excluded from the study. Endocervical smears and cultures were examined for the presence of Neisseria gonorrhoeae. 46% of the 100 patients had gonococcal infections. Since the cultures usually fail to detect all cases of gonococcal infection, the prevalence of gonorrhea among PID patients was probably somewhat higher than the findings indicated. Women with gonococcal PID, when compared to the nongonococcal PID patients tended to be younger and to have more severe PID and a history of 2 or more previous episodes of PID. The gonococcal and nongonococcal PID patients did not differ in marital status and in the number of sexual partners they had. Given the high incidence of gonococcal infection among PID patients in Lusaka, the recommendation was made that PID patients should routinely be treated with an antibiotic regimen of sufficient strength to counter gonococcal infection. Study findings were presented in tabular form.

Sexually transmitted diseases in Lusaka.

This is a preliminary epidemiological report on sexually transmitted diseases in Zambia. An analysis of data derived from a sample study of 1,000 consecutive female and 5,000 male S.T.D. patients seen at the University Teaching Hospital in Lusaka, is presented. The predominant diseases and the prevailing socio-economic, cultural and other factors responsible for the high prevalence of S.T.D. in Zambia are discussed.