Lymphoid cells mediating tumor-specific cytotoxicity to carcinoma of the urinary bladder. Separation of the effector population using a surface marker.

Peripheral lymphocytes from patients with urinary bladder carcinoma and controls have been separated on the basis of rosette formation with sheep erythrocytes. The fractions were tested for tumor-specific cytotoxicity. The E rosette-forming cells of purity 90% respond well in PHA-induced cytotoxicity but are totally inactive in the tumor assay. The non-E rosette-forming cells (purity 91%) give enhanced activity in the tumor-specific cytotoxicity as well as in antibody-mediated target cell lysis in a model system. These data support the notion that the effector cells in cell-mediated immunity to carcinoma of the urinary bladder are members of the nonthymus-derived population of peripheral lymphocytes.

Are radiographers an influencing factor in the radiation protection practices of speech-language therapists performing videofluoroscopic swallowing studies?

INTRODUCTION: A videofluoroscopic swallowing study (VFSS) is a fluoroscopic examination conducted by radiographers and speech-language therapists (SLTs) to assess dysphagia. Given the potential of SLTs to feed patients during the procedure, they may be exposed to radiation. The research aimed to assess radiation protection practices utilised by SLTs to determine if radiographers have a role in providing ongoing practical education. METHODS: An online questionnaire was distributed to SLTs from six countries (Australia, Canada, Ireland, New Zealand, United Kingdom and United States of America). Responses were analysed quantitatively using frequencies and chi-square analysis (p = 0.05) and supported by written comments. RESULTS: A total of 224 responses were analysed. Thyroid shields (94%) were used more frequently than full aprons (72%). Differences (p < 0.0001) were seen between Australian and USA participants regarding the use and position of radiation monitors; 43% of Australian participants stating they always used a monitor, compared to 75% of USA participants. Nearly all Australian SLTs wore monitors under shielding (92%) and at waist level (69%), while USA participants reported wearing them outside shielding (97%) and at thyroid level (94%). Participants' radiation practice was influenced primarily by other SLTs (64%), followed by radiographers (57%). However, written comments revealed the significance of the radiographer in providing training as "radiographers are excellent at ensuring we [use] right equipment, stand in the right places and use exposure monitoring". CONCLUSION: SLTs did not always adopt the ICRP principle of shielding and there were inconsistencies with regards to the use and placement of radiation monitors. Radiographers are well positioned to provide advice with regards to safe practice. IMPLICATIONS FOR PRACTICE: Opportunities to enhance radiation protection practices are evident, as is the advising role of radiographers.

Metallothionein messenger RNA regulation in the mottled mouse and Menkes kinky hair syndrome.

Menkes kinky hair syndrome is an X-linked neurodegenerative disorder, causing tissue-specific increases in copper and metallothionein content. A mouse model is provided by hemizygotes for mutant alleles at the X-linked mottled locus. Herein we test the possibility that the primary defect in both species is in metallothionein gene regulation. We show that metallothionein-I messenger RNA (mRNA) (mouse) and metallothionein-II mRNA (human) are elevated in mutant fibroblasts. However, comparable dose-response curves in mutant and control cells are generated when mouse metallothionein-I mRNA concentrations are measured in cells exposed to varying concentrations of cadmium or copper (metallothionein inducers). Furthermore, when mutant and control cells are grown to achieve overlapping intracellular copper concentrations in the two cell types, metallothionein-I (mouse) and metallothionein-II (human) mRNA levels are proportional to the intracellular copper concentrations. Finally, in paired determinations in blotchy hemizygote and littermate kidneys containing comparable copper levels, metallothionein-I mRNA contents are very similar. The observations suggest that elevated intracellular copper in these mutants induces metallothionein synthesis by normal regulatory mechanisms.

Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction.

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.

Anti-squamous tumor antibodies in patients with squamous cell carcinoma.

Patients with advanced squamous cell carcinomas were shown to have serum antibodies directed towards cultured squamous tumor cells as shown by quantitative membrane immunofluorescence. The sera of these same patients did not react with a variety of other cultured tumor cells. Serum obtained from normals or from patients with other forms of cancer (transitional cell carcinoma, adenocarcinoma, and melanoma) did not give positive reactions. When the sera of squamous carcinoma patients were chromatographed on Sephadex G-150, tumor-reactive antibodies were recovered solely in the 19 S fraction, suggesting immunoglobulin M as the immunoglobulin isotype involved. Identification of the squamous tumor cell-reactive immunoglobulin as ijmunoglobulin M was confirmed by quantitative immunofluorescence with the use of class monospecific antisera to human immunoglobulins.

Clinical status and rate of recovery of blood lymphocyte levels after radiotherapy for bladder cancer.

Peripheral blood lymphocytes and leukocyte levels were monitored in 34 patients with bladder carcinoma before, during, and up to 5 years after radiotherapy. Radiotherapy in doses 6500 to 8500 rads caused a marked decline in the numbers of circulating leukocytes and particularly lymphocytes. In patients clinically free of disease for 5 years, lymphocyte counts returned to pretherapy levels within 3 years after radiotherapy. In contrast, in patients with recurrent or residual tumors lymphocyte counts failed to reach pretherapy levels within 3 years after therapy. The rate of recovery from radiation-induced lymphopenia was significantly different for patients who were free of disease as compared to those with recurrent or residual tumor (p less than 0.05). No correlation was found between posttherapy leukocyte levels and clinical status.

A 7-year analysis of anti-Gag (p17 and p24) antibodies in HIV-1-seropositive patients with haemophilia: immunoglobulin G titre and avidity are early predictors of clinical course.

OBJECTIVE: To study the humoral immune response to HIV-1 p17 and p24 Gag proteins longitudinally and assess any prognostic value. DESIGN AND METHODS: Fifteen HIV-1 infected patients with haemophilia were asymptomatic at entry to the study in 1986. Each patient was monitored at 6- to 12-month intervals for up to 7 years for p17 and p24 immunoglobulin (Ig)G titres, p17 IgG avidity, total IgG, p24 antigenaemia and CD4 cell counts. Results were correlated with the clinical course. RESULTS: Between 1986 and 1993, six of the patients developed CD4 cell counts below 200 x 10(6)/l (AIDS patients) while nine retained counts above this level (asymptomatic patients). All AIDS patients were characterized by declining IgG titres to p17 and p24 from 1986-1987 onwards. A reduction in specific p17 and p24 IgG preceded, by at least 3-4 years, the onset of CD4 cell depletion (< 200 x 10(6)/l). These six patients also had significantly lower p17 IgG avidity than the asymptomatic patients throughout the study. CONCLUSION: Titres of p17 and p24 IgG appeared to alter in the same manner. A progressive reduction in IgG titres and a low p17 IgG avidity were earlier predictors of disease progression than CD4 cell counts or p24 antigenaemia.

Cryopreservation of immunological memory and other lymphoid cell functions.

The following components of the immune response by mouse spleen cells were retained after cryopreservation at --110 degrees C: direct and indirect plaque formation, avidity of antibody secreted by plaque forming cells, and induction of primary or secondary adoptive transfer responses. Human peripheral blood lymphocytes retained the capacity to mediate antibody-dependent cytotoxicity and cell mediated immunity to tumor in vitro.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers.

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Detection of HLA antigens on lymphoblastoid and epithelial cell lines and cross-reactivity of HLA-Cw5 and HLA-Cw8.

Antibody-dependent cell-mediated cytotoxicity was used to detect HLA antigens on tissue cultured lymphoblastoid cells (phytohemagglutin blasts and Epstein-Barr virus lines) and transitional cell carcinomas. The results agreed with those obtained on fresh peripheral blood lymphocytes by conventional HLA typing. The same HLA antigens were detected on cells from an individual irrespective of their tissue origin or length of time in vitro. Antibody-dependent cell-mediated cytotoxicity (ADCC) showed that HLA-Cw5 and HLA-Cw8 were cross-reactive. An HLA-Cw5 antiserum that was negative for HLA-Cw8 positive cells in complement-mediated lymphocytotoxicity reacted strongly with HLA-Cw8 donor cells in ADCC. Similarly HLA-Cw8 antibodies were detected in HLA-B14 antisera, which reacted on all HLA-Cw5-positive donor cells. Absorption of sera with HLA-Cw5-positive lymphoid cells removed HLA-Cw5 and HLA-Cw8 specificities but spared HLA-B14. Absorption of HLA-B14 antisera with HLA-B14/Cw8-positive cells removed HLA-Cw5, HLA-Cw8, and HLA-B14 reactivities. Sequential immune precipitation and gel electrophoresis confirmed that HLA-Cw5 and HLA-Cw8 were cross-reactive and that HLA-B14 was physically separable from HLA-Cw8.

Decline in CTL and antibody responses to HIV-1 p17 and p24 antigens in HIV-1-infected hemophiliacs irrespective of disease progression. A 5-year follow-up study.

CTL and antibody responses to HIV-1 p17 and p24 antigens were monitored from 1986-1991, in 4 hemophiliacs. The patients had been infected with HIV-1 between 1980 and 1984. Two patients have remained asymptomatic while two progressed to AIDS in 1990. CTL were boosted by culturing with peptides from p17 aa 86-115, or p24 aa 265-279; and aa 270-373 or PHA. Lysis was measured on autologous or allogeneic targets pulsed with peptides or infected with recombinant vaccinia virus carrying HIV-1 gag or influenza A matrix genes. Antibodies to p17 and p24 were tested by ELISA using peptides and by Western blotting. High levels of CTL activity to p17 and p24 antigens could be generated only with lymphocytes from the two asymptomatic patients between 1986 and 1989, but these responses were absent in 1990 and 1991. Antibodies to p17 peptides disappeared in parallel with CTL activity. Antibodies to some p24 peptides also declined but most patients retained activity to others. In all patients a > or = 3-fold increase in CD8+ cell numbers occurred over time and accompanied the decline of CTL and antibody responses. The loss of CTL and p17 antibodies occurred irrespective of whether patients remained asymptomatic or progressed to AIDS in the intervening two years.

Effect of direct injection of melanin-concentrating hormone into the paraventricular nucleus: further evidence for a stimulatory role in the adrenal axis via SLC-1.

Melanin-concentrating hormone (MCH) is implicated in the control of a number of hormonal axes including the hypothalamic-pituitary adrenal (HPA) axis. Previous studies have shown that there is evidence for both a stimulatory and an inhibitory action on the HPA axis; therefore, we attempted to further characterize the effects of MCH on this axis. Intracerebroventricular injection of MCH increased circulating adrenocorticotropic hormone (ACTH) at 10 min post injection. Injection of MCH directly into the paraventricular nucleus (PVN) was found to increase both circulating ACTH and corticosterone 10 min after injection. Additionally, MCH was found to increase corticotropin-releasing factor (CRF) release from hypothalamic explants, and this effect was abolished by the specific SLC-1 antagonist SB-568849. Neuropeptide EI, a peptide from the same precursor as MCH was also found to increase CRF release from explants. These results suggest that MCH has a stimulatory role in the HPA axis via SLC-1, and that MCH exerts its effects predominantly through the PVN CRF neuronal populations

Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro.

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.

Absence of association between Alzheimer disease and the -491 regulatory region polymorphism of APOE.

A novel polymorphism (-491 A/T) within the regulatory region on the apolipoprotein E gene has recently been reported to be associated with risk for Alzheimer disease (AD). To test this association in an independent data set, we have examined this polymorphism in a sample of 88 well-characterized AD cases and compared the allele frequency and genotype frequencies for this polymorphism with those observed in 112 cognitively normal subjects drawn from the same ethnic group. These results suggest that in the current data set at least, the -491 A/T polymorphism is not associated with risk for AD, but may be in partial linkage disequilibrium with the APOE epsilon2/epsilon3/epsilon4 polymorphism.

Digital imaging colposcopy: corrected area measurements using shape-from-shading.

The quantitative measurement of areas on the cervix is of interest to researchers studying the natural history of human papilloma viral lesions. Measurement of areas from images obtained through a colposcope are, however, inherently in error due to the image being a two-dimensional projection of a three-dimensional object. The ability to correct for these errors through use of digital imaging colposcopy and a practical application of a shape from shading algorithm was developed in this study. The shape from shading technique requires empirical measurement of the relationship between observed light intensity and the viewing angle (referred to as a reflection map). It was found that a population mean reflection map provided a correction that was about as accurate as using an individual's own reflection map (making it unnecessary to measure a map for each exam). Digital red filtering of the images increased accuracy and precision of measurement.

The cognitive assessment screening test (CAST) for dementia.

The purpose of this study was to assess the usefulness, the sensitivity, and the specificity of the Cognitive Assessment. Screening Test (CAST), a paper-and-pencil self-administered cognitive test designed to screen elderly people for possible dementia, for use in general physicians' offices, requiring little expertise or staff time. CAST consists of three parts: part A (relatively easy), part B (more demanding), and part C (self-report of concerns). CAST was administered in two studies to: (1) 19 patients known to be mildly to moderately demented versus 24 age-matched normal controls (to establish cutoff standards); and (2) a "real world" sample of 26 elderly patients not known to be demented, attending a general medicine clinic. The sensitivity and specificity of CAST were compared with the Mini-Mental State Examination (MMSE) and the Blessed Dementia Scale cognitive portion (BDS-cog). In study 1, controls were given a detailed neuropsychological battery; in study 2, all patients were given the neuropsychological battery, which served as the "gold standard" to identify individuals with cognitive impairment. In study 1, the cutoff scores for dementia using CAST (Parts A and B) were established. CAST discriminated demented patients from controls with a sensitivity of 95% and a specificity of 88%; the MMSE had a sensitivity of 74% and a specificity of 100%; and the BDS-cog had a sensitivity of 100% and a specificity of 96%. In study 2, CAST discriminated cognitive impairment with a sensitivity of 88% and a specificity of 100%, the MMSE had a sensitivity of 38% and a specificity of 100%; and the BDS-cog had a sensitivity of 50% and a specificity of 94%. Part C was not used to discriminate demented from normal elderly individuals, but to screen for those concerned about their cognitive functioning. CAST is highly useful as a dementia screening test, with sensitivity and specificity equal to or better than the MMSE and BDS-cog, yet requiring minimal examiner time and little training or experience to administer.

Cadmium, zinc, and copper metabolism in the mottled mouse, an animal model for Menkes' kinky hair syndrome.

Studies of uptake and release of 64Cu, 109Cd, and 65Zn in suckling C57BL/6J male mice revealed kinetics and distributions that differed for each metal both within and among the organs analyzed, suggesting distinct, albeit overlapping, mechanisms for transport and binding of each metal. In mutants, there were tissue-specific increases in copper-binding capacity. In hemizygotes (Moblo/y) accumulation of 64Cu was increased in kidney, lung, and duodenum. In heterozygotes (Moblo/+), 64Cu content was increased in kidney, with a smaller increase in lung, and no change in duodenal Cu. Decreased 64Cu accumulation was seen in liver in both hemi- and heterozygotes. In contrast, 64Zn and 109Cd accumulation in organs of heterozygote mice was not significantly distinguishable from normal. In skin and connective tissues there is excessive accumulation of 64Cu in Moblo/+ and Moblo/y, no abnormality in heterozygote 65Zn accumulation, but a clear decrease in heterozygote 109Cd content. In both mutant kidney and liver, there was an aberrant subcellular distribution of 64Cu, with the major fraction of sequestered 64Cu in the cytosol. Our studies establish that in spite of the ubiquity of metallothioneins and the structural similarities of those that have been characterized, there is specificity and functional heterogeneity in metal binding among tissues. The aggregate data suggest that there are unique regulatory mechanisms for the metabolism of copper and zinc, while there exists, in part, an inverse relationship between the binding of copper and cadmium. Our data further suggest that the blotchy mutation involves a specific cytosolic copper storage or transport protein also capable of binding cadmium.

Assessment of the relative cytotoxicity of Porphyromonas gingivalis cells, products, and components on human epithelial cell lines.

Established human cell lines derived from a transitional cell carcinoma (J82), a squamous carcinoma (SCaBER), and a normal urothelium (HCV-29) were used to assess the relative cytotoxicity and tissue specificity of putative virulence determinants of P. gingivalis W83. Intact cells of W83 had no effect on any of the cell lines, whereas disrupted cells caused extensive cytotoxicity particularly to monolayers of HCV-29 and J82. The purified cysteine proteinase, gingivain, caused marked disruption of the basement membrane of the SCaBER monolayers but had no cytotoxic effects. Use of the thiol-inhibitor, 2,2'-dipyridyl disulphide revealed that the effects observed with the vesicles and the culture supernatant were due to the presence of the cysteine proteinase. The attachment of vesicles to the SCaBER cells was evident in electron micrographs. Short-chain volatile fatty acids added in concentrations equivalent to those present in the culture supernatant had no effect on any of the cell lines tested. Culture supernatants obtained from high speed centrifugation (150,000 x g) showed no cytotoxic effects. This was in marked contrast to the supernatant obtained by lower sedimentation (18,000 x g), which damaged all monolayers tested. These results suggest that these cell lines are potentially useful for assessing putative virulence determinants of P. gingivalis and other periodontal pathogens.

Vocal cord dysfunction presenting as asthma.

A 14 year old boy presented with deteriorating asthma and marked stridor. Neither asthma nor stridor responded to an increase in anti-asthma medication, including high dose oral steroids. Indirect laryngoscopy revealed adduction of the vocal cords throughout the respiratory cycle, a phenomenon previously identified as a psychosomatic conversion reaction. When gently confronted with these findings and offered psychological assistance the boy's symptoms abated totally. After two sessions of hypnotherapy he has had better control of both his physical (asthma) and psychological problems.

Nursing directors' perceptions of the dental components of the Minimum Data Set (MDS) in nursing homes.

The Directors of Nursing (DON's) of all 428 nursing homes in the state of Iowa were sent a pre-tested questionnaire. The aim of the study was to examine the perceptions by nursing directors on the utility of a dental component of the Minimum Data Set (MDS) in identifying residents with dental problems in their facility. The return rate was 55.1% from the DON's. It was reported that 66.4% of the homes had a training program for the MDS and that in 38.0% of the homes there was only one person doing the assessments. The majority (76.0%) of the DON's stated that the MDS was useful in tracking residents and that it did help them to identify dental problems. When asked how often it was useful in the identification of dental needs, only 9% stated that it was often useful. Also, regarding frequency of dental appointments for the residents, a mean of 51.0% of residents were estimated to have received some dental care during the previous year (means of 33.2% at the facility, 22.4% at a dental office, and 0.4% in a hospital). If change is to occur, the dental profession must try to get the nursing home assessors to convince the nursing homes to use the oral/nutritional/dental sections of the MDS as they were intended under the OBRA regulations.