SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for this function and TNF-induced cathepsin G is a candidate target.

The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNF alpha-mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNF alpha-induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNF alpha but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNF alpha. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.

Immunoreactivity of recombinant squamous cell carcinoma antigen and leupin/SCCA-2: implications for tumor marker detection.

OBJECTIVES: Squamous cell carcinoma antigen (SCCA) is a member of the serpin superfamily, and has been used as a serological tumor marker for cervical squamous cell carcinomas. We have identified a closely related serpin gene, leupin (SCCA-2), which may be the fraction previously thought to be the acidic isoform of SCCA. The purpose of this study is to isolate the individual recombinant proteins, to examine their reactivity with current immunological detection methods, and to use a gene-specific method to examine their expression in the uterine cervix. METHODS: We have expressed and purified recombinant forms of SCCA and leupin individually. The proteins were characterized with respect to their isoelecric points and their reactivity with the monoclonal antibody from the current tumor marker diagnostic immunoassay (IMx SCC). Reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers was used to examine expression of both genes. RESULTS: Isoelectric focusing shows that leupin is the more acidic antigen with a determined pI for recombinant leupin (rLeupin) of 6.01, with rSCCA having a pI of 6.17. The IMx SCC monoclonal antibody recognized both rSCCA and rLeupin in immunoassays and immunoblots and both genes are expressed in normal cervix and in cervical carcinoma tissue. CONCLUSIONS: The findings from this study suggest that all previous clinical studies examining SCCA expression have used methodology that detects two gene products. The confirmation that leupin or SCCA-2 is the more acidic protein and that its expression is significantly elevated in cervical cancer suggests that this gene product may be the more important tumor marker.

The expanding superfamily of serpins: searching for the real targets.

The serpin superfamily of proteins has expanded rapidly in recent years as represented by the ovalbumin-type serpin subfamily. PCR methods have been used to identify new ovalbumin-type serpins, including leupin or SCCA-2, a close relative of SCCA. Although in vitro inhibition of proteases by these serpins has been shown, and we have evidence that leupin can protect cells against apoptosis, the exact role of these serpins is not well understood. Finding relevant targets is a major challenge in serpin biology, and we have investigated the yeast-2-hybrid trap for identification of new interactions. Preliminary studies suggest that serpins are at the upper limit for bait size, but this technique may be useful for identifying interactions where full-length serpin protein is not required.

Transfusion-transmitted babesiosis in Washington State: first reported case caused by a WA1-type parasite.

Most cases of babesiosis reported in the United States have been tickborne and caused by Babesia microti, the etiologic agent of all previously described transfusion-transmitted cases. A 76-year-old man with the first recognized case of transfusion-transmitted infection with the recently identified WA1-type Babesia parasite is described. The subject received multiple blood transfusions in 1994. Indirect immunofluorescent antibody testing of serum from 57 blood donors implicated a 34-year-old man (WA1 titer, 1:65,536) whose donation had been used for packed red cells. Isolates of the organisms that infected the recipient and the donor, both of whom were spleen-intact residents of Washington State, were obtained by hamster inoculation. The DNA sequence of a 536-bp region of the nuclear small subunit-rRNA gene of both isolates was identical to that of WA1 (isolated in 1991 from the index WA1 case-patient). Effective measures for preventing transmission of babesiosis by blood transfusion are needed.

Maintenance electroconvulsive therapy and cognitive function.

BACKGROUND: ECT is rarely used as a prophylactic treatment. A 74-year-old woman with unstable bipolar affective disorder receiving maintenance ECT presented a unique opportunity to measure the cognitive effects of continuing ECT. METHOD: A single case report with serial psychometric testing during over 400 ECT treatments as a single maintenance treatment. RESULTS: Serial testing did not demonstrate progressive cognitive deterioration, but consistent cognitive deficits typical of acute treatment were evident. The degree of cognitive difficulty may be related to the frequency of treatment. CONCLUSIONS: Maintenance ECT can be an effective prophylactic treatment for selected patients. Cognitive effects would appear to be no greater than with acute treatment and seem to be non-progressive.

Trends in the use of accident and emergency services by the homeless on the Wirral.

OBJECTIVE: To examine the use of accident and emergency (A&E) services by the homeless, with particular reference to the use of psychiatric services generated by this attendance. METHODS: A retrospective study of attendance registers at a large A&E department over an eight year period, during which there were 566 separate attendances by homeless patients out of a total of 421 237 adult attendances (1.3 per 1000). RESULTS: There was no significant change in rates of attendances over the eight year period (P = 0.41), in the broad demographic details of the groups, or in the use of psychiatric services. CONCLUSIONS: There has been no significant increase in the use of A&E services by homeless people in the population studied over the eight year period. Several possible explanations for this are given.

Methylxanthines with adenosine alter TNF alpha-primed PMN activation.

Methylxanthines are best known as phosphodiesterase inhibitors that cause a rise in intracellular cAMP. One would expect the two methylxanthines, caffeine and pentoxifylline, to have similar actions on neutrophils (PMN). However, caffeine stimulated and pentoxifylline inhibited PMN oxidative activity. Micromolar concentrations of pentoxifylline decreased native and recombinant tumor necrosis factor-alpha (TNF alpha)-primed formyl met-leu-phe (fMLP)-stimulated PMN chemiluminescence, superoxide production and myeloperoxidase (MPO) release. In contrast, equal concentrations of caffeine increased chemiluminescence and MPO release with no effect on superoxide production. These activities of the methylxanthines were only observed in the presence of physiological concentrations of adenosine, and were abolished by the treatment of the PMN with adenosine deaminase. The activities of adenosine, pentoxifylline and caffeine on PMN activity could not be readily explained by changes in PMN [cAMP]. Thus for TNF alpha-primed PMN, pentoxifylline decreases PMN activity by enhancing the effect of adenosine on degranulation and superoxide production; whereas caffeine increases PMN activity by counteracting the effect of adenosine on degranulation.

Identification of a novel human serpin gene; cloning sequencing and expression of leupin.

A novel serpin gene has been isolated, cloned and sequenced. A PCR amplified fragment of the gene was originally identified from human genomic DNA, and the full-length cDNA was subsequently isolated from HeLa cells and sequenced. The novel serpin is very high in protein sequence similarity (91.8%) to the squamous cell carcinoma antigen (SCCA), but contains substantial differences in the reactive site loop sequence, including a different amino acid (leucine) in the P1 position. The gene product, named leupin, is expressed in HeLa cells, SKGIIIa cells and human placenta. The protein has a predicted M(r) of 44,857 and an isoelectric point of 6.04 which is consistent with the more acidic form of SCCA associated with squamous cell carcinomas.

Culture-confirmed pneumonia due to Chlamydia pneumoniae.

Diagnosis of infection caused by Chlamydia pneumoniae, a newly recognized respiratory pathogen, has proved difficult. Between July 1987 and April 1988, culture and serologic tests for C. pneumoniae were done on specimens from 49 patients with pneumonia seen at an Atlanta hospital emergency room. Cultures from 3 patients (6%) grew C. pneumoniae. Genus-specific Chlamydia complement fixation titers and microimmunofluorescence titers for C. pneumoniae were suggestive of acute infection in all 3 culture-positive patients. Three other patients had evidence of acute disease by published criteria for antibody titers. Most studies of C. pneumonia have not had culture-proven cases; the 6% rate of positive cultures in this study support the role of C. pneumoniae as a cause of pneumonia. More widespread availability of simplified culture systems for C. pneumoniae is needed. Caution should be used when interpreting serologic tests in the absence of culture confirmation.

Erythromycin for persistent or recurrent nongonococcal urethritis. A randomized, placebo-controlled trial.

OBJECTIVE: To evaluate the efficacy of a 3-week regimen of erythromycin for treatment of persistent or recurrent nongonococcal urethritis in men. DESIGN: A prospective, randomized, double-blind trial with follow-up at 2, 4, and 8 weeks after enrollment. PATIENTS: Seventy-seven evaluable men with objective evidence of nongonococcal urethritis (36 in the erythromycin group and 41 in the placebo group) with a mean age of 28 years, a median duration of urethritis of 3 months, and a median number of three previous antimicrobial regimens. INTERVENTION: Erythromycin, 500 mg, or placebo four times daily for 3 weeks. RESULTS: After 2 weeks of treatment, urethral symptoms resolved in 13 of 25 erythromycin-treated patients compared with 8 of 34 placebo-treated patients (P = 0.03). Erythromycin also resulted in more frequent resolution of urethral discharge and leukocytosis at all visits compared with placebo but these differences were not statistically significant. First-voided urine leukocyte counts decreased, however, by a median of 89% (95% CI, -96% to -67%) in the erythromycin group compared with 23% (CI, -73% to 83%) in the placebo group after treatment (P = 0.02 for the difference in changes). Further, in men with prostatic inflammation, urinary leukocyte counts decreased by a median of 94% (CI, -99% to -83%) after treatment in erythromycin-treated patients compared with a 46% increase (CI, -57% to 290%) in placebo-treated patients (P = 0.0003 for the difference in changes). CONCLUSION: A 3-week regimen of erythromycin was more effective than placebo in improving symptoms and in reducing pyuria in men with persistent or recurrent nongonococcal urethritis, especially among men with prostatic inflammation.

Treatment of Chlamydia trachomatis genital infection.

Chlamydia trachomatis infections are the most frequent bacterial sexually transmitted diseases in the United States, with an estimated 4 million cases occurring annually. The mainstay of treatment for chlamydial infections has been the tetracyclines. Doxycycline, comparable in cost to tetracycline and with a less frequent dosage schedule, is the drug of first choice. Erythromycins are the drugs of choice for infections in pregnant women, nursing mothers, infants, children, and adults unable to tolerate tetracyclines. beta-Lactam antibiotics have had variable efficacy against C. trachomatis infections, although recent studies suggest that amoxicillin may be an effective alternative for C. trachomatis infection during pregnancy. Quinolones are currently being tested and may be used in alternative regimens in the future. The effectiveness of current antimicrobial regimens in preventing long-term complications of chlamydial infections should be determined in research studies.

Chlamydia trachomatis infection of human fallopian tube organ cultures.

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.

Seroepidemiology of chlamydia in Costa Rica.

A population-based study of the sero-epidemiology of chlamydia was performed among a nationally representative sample of 760 Costa Rican women aged 25 to 59 years. Interviews and sera collection were completed between September 1984 and February 1985. The overall seroprevalence of chlamydial antibodies among these women was 56.1%. Women 25 to 39 years of age had a seroprevalence of 51.1%, while women 40 to 59 years of age had a seroprevalence of 64.2%. Women who reported no prior sexual activity had a seroprevalence rate of 48.6%, compared with a seroprevalence rate of 80.7% among women who reported three or more lifetime sexual partners. The geometric mean titre (GMT) of seropositive women ranged from 34.4 among the women who reported no prior sexual activity to 155.0 among the women with three or more lifetime sexual partners. Sero-positivity was more consistently associated with sexual activity than with age. Women with serological evidence of past Herpes simplex virus type 2 (HSV-2) or syphilis infection were more likely to be seropositive than were women without evidence of exposure to these sexually transmitted diseases, even when controlled for age and the number of lifetime sexual partners. The seropositivity among never sexually active women indicates the probable presence of Chlamydia pneumoniae infections, while the high seroprevalence of chlamydial antibodies among the sexually active women suggests that sexually transmitted Chlamydia trachomatis infections represent a public health problem not previously quantified in Costa Rica. Further seroepidemiological and/or culture studies are warranted to determine the incidence and prevalence of sexually transmitted chlamydial infection among men and younger women.

Quantitative culture of endocervical Chlamydia trachomatis.

We examined the number of Chlamydia trachomatis inclusions produced in the initial passage of cell cultures of endocervical specimens from 1,231 women with positive chlamydial cultures who attended a sexually transmitted diseases clinic. Youth, white race, oral contraceptive use, and concurrent infection by Neisseria gonorrhoeae were associated with high chlamydial inclusion counts. Youth, white race, and oral contraceptive use were independent determinants of a high chlamydial inclusion count in women without concurrent gonorrhea but not in women with gonorrhea. Results of our study suggest that the degree of chlamydial excretion from the infected cervix may be influenced by characteristics of the patient being tested and may affect the ability to detect C. trachomatis in different patient groups.

PIP: The relationships between selected epidemiological variables and the number of organisms detected in 1st passage in cell culture of specimens obtained from patients at an Indiana sexually transmitted disease clinic who were infected with Chlamydia trachomatis was investigated. Endocervical C trachomatis was detected in the initial passage of cell culture in 1300 (25%) of the 5276 eligible women. 599 (46%) of these infected women were also infected with Neisseria gonorrhoeae. 1769 (34%) were oral contraceptive (OC) users and 780 (60%) were black. Inclusion count data were obtained for only 1231 chlamydia-infected women given the need to exclude pregnant women and IUD users. The inclusion count distribution was as follows: less than or equal to 100 IFU/ml, 25%; 101-1000 IFU/ml, 40%; 1001-10,000 IFU/ml, 21%; and over 10,000 IFU/ml, 14%. Multivariate analysis of these counts identified young age (under 20 years), current OC use, and concurrent gonorrhea as the most significant risk factors for endocervical C trachomatis. The cervical signs of ectopy, mucopus, and friability were also associated with chlamydial infection. Among women with gonorrhea, only concurrent trichomoniasis was associated with the inclusion count. A history of prior sexually transmitted diseases or the presence of concurrent infection with trichomoniasis were associated with lower inclusion counts. Since the degree of detectability of chlamydial excretion may be affected by certain patient characteristics such as those identified in this study, possible bias in chlamydial detection tests must be considered.

Effect of patient characteristics on performance of an enzyme immunoassay for detecting cervical Chlamydia trachomatis infection.

We compared the performance of a commercial enzyme immunoassay (EIA) (Chlamydiazyme; Abbott Diagnostics, North Chicago, Ill.) with that of cell culture for the detection of Chlamydia trachomatis cervical infection in 1,417 women attending public health clinics. Confirmatory chlamydial testing by a direct fluorescent-antibody test (MicroTrak; Syva Co., Palo Alto, Calif.) was performed on specimens from women who had positive EIAs. Overall, only 57% of women who had a positive chlamydial test by cell culture were also positive by EIA. We noted a strong association between the number of chlamydial inclusions in cell culture and a positive EIA outcome. The proportion of culture-positive women who also had a positive EIA declined with age and a history of previous sexually transmitted disease and increased among oral contraceptive users. The results of direct fluorescent-antibody confirmatory testing suggested that cell culture was also insensitive for the detection of C. trachomatis infection. Our observations demonstrate that the performance of the chlamydial EIA may vary greatly with individual patient characteristics and that the utility of EIA as a screening test may be limited, especially in older women.

Enzyme immunoassay to determine exposure to Chlamydia pneumoniae (strain TWAR).

Recent studies suggest that a group of Chlamydia strains known as TWAR, which are now proposed to be a new species called Chlamydia pneumoniae, may be a frequent cause of respiratory disease in the United States and many other countries. Current serotesting methods do not allow rapid screening of large numbers of samples to distinguish C. trachomatis exposure from C. pneumoniae exposure. We developed an enzyme immunoassay to decrease cross-reactivity between immunoglobulin G antibodies reactive with C. trachomatis and C. pneumoniae. Elementary bodies of C. trachomatis or C. pneumoniae were treated with a detergent-chelating solution to decrease the reactivity of the common lipopolysaccharide antigens. Sera from four groups of patients, totaling 143 persons, were tested by this assay. The prevalences of titers of greater than or equal to 128 to C. trachomatis and C. pneumoniae, respectively, were as follows: (i) for 23 women seropositive for C. trachomatis by the microimmunofluorescence test, 21 (91%) and 18 (78%); (ii) for 50 adult blood donors, 13 (26%) and 39 (78%); (iii) for 40 sexually transmitted disease clinic patients, 20 (50%) and 32 (80%); (iv) for 30 healthy children 5 to 7 years old, 0 (0%) and 8 (27%). Western blots (immunoblots) of each antigen corroborated the differential reactivity of C. trachomatis-positive, C. pneumoniae-negative and C. trachomatis-negative, C. pneumoniae-positive serum samples. Western blots of serum samples from rabbits immunized with either C. trachomatis or C. pneumoniae elementary bodies revealed at least two protein bands (30 and 80 kilodaltons) which appeared to represent unique C. pneumoniae antigens.

Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis.

We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle.