Consistent FFP2-masking as part of reducing viral respiratory infections on medical wards for allogeneic hematopoietic stem cell transplantation.

Patients undergoing allogenic hematopoietic stem cell transplantation (allo-HSCT) are highly susceptible to infections. The consequent use of masks on wards for allo-HSCT has been controversial in the past decades and was not common before the COVID-19 pandemic. We retrospectively compared incidence and outcomes of viral respiratory infections during allo-HSCT on our specialized ward between 01/2018 and 09/2020 to the era of FFP2 masking between 10/2020 and 10/2022 covering similar seasons of the year. Each group consisted of 150 matched patients. The usage of FFP2 masks reduced the incidence of viral respiratory infections from 22.1 to 2.1% (p < 0.005). This reduced the time on ward from a median of 26 days to 23.5 days (p = 0.002). It also resulted in less use of CT-scans (p = 0.003) and bronchoalveolar lavage procedures (p = 0.057). Median time to proof of infection was 21 days after admission in both groups. No difference was detected in progression free survival, hospital survival or non-relapse mortality (p = 0.78). Our retrospective results indicate that FFP2 masks worn by patients and hospital staff may help to significantly reduce the incidence of viral respiratory infections, including COVID-19, shorten the in-hospital time, and reduce costs without affecting survival.

Human kallikrein gene 13 (KLK13) expression by quantitative RT-PCR: an independent indicator of favourable prognosis in breast cancer.

Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase-polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival (P<0.001 and P=0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I-II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 (P=0.001) and 0.24 (P=0.008), respectively, for the Grade I-II group; 0.36 (P=0.008) and 0.44 (P=0.038), respectively, for the node positive group and 0.36 (P=0.008) and 0.18 (P=0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 (P=0.012). For patients in the node positive and oestrogen receptor positive subgroup (n=51) the adjusted hazard ratio was 0.25 (P=0.006) and for the node positive and progesterone receptor positive subgroup (n=46) the hazard ratio was 0.24 (P=0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase-polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma.

Do wine polyphenols modulate p53 gene expression in human cancer cell lines?

BACKGROUND: The p53 gene is an established tumor suppressor and an inducer of apoptosis. We here attempt to determine whether the putative anticarcinogenic properties attributed to red wine and its polyphenolic constituents depend, at least in part, upon their ability to modulate p53 expression in cancer cells. METHODS: Three human breast cancer cell lines (MCF-7, T47D; MDA-MB-486) and one human colon cancer cell line [Colo 320 HSR (+)] were treated for 24-h with each of four polyphenols [quercetin; (+)-catechin, trans-resveratrol; caffeic acid] at concentrations ranging from 10(-7) M to 10(-4) M, after which, p53 concentrations were measured in cell lysates by a time-resolved fluorescence immunoassay. RESULTS: None of the polyphenols tested affected p53 expression in the breast cancer cell lines T-47D and MDA-MB-486. p53 content of MCF-7 breast cancer cells (wild-type) was increased by caffeic acid, decreased by resveratrol, and showed a twofold increase with catechin, that reached borderline statistical significance; however, none of these effects were dose-responsive. Colo 320 HSR (+) cells (with a mutant p53 gene) had lower p53 content upon stimulation, reaching borderline statistical significance, but without being dose-responsive, in the presence of caffeic acid and resveratrol. Apart from toxicity at 10(-4) M, quercetin had no effect upon these four cell lines. CONCLUSIONS: The observed p53 concentration changes upon stimulation by polyphenols are relatively small, do not follow a uniform pattern in the four cell lines tested, and do not exhibit a dose-response effect. For these reasons, we speculate that the putative anticarcinogenic properties of wine polyphenols are unlikely to be mediated by modulation of p53 gene expression.

Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues.

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.

Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.

BACKGROUND: The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein. METHODS: Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6. RESULTS: The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 microg/L and upper concentration range of 200 microg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form. CONCLUSIONS: This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.

Is ICI 182,780 an antiprogestin in addition to being an antiestrogen?

The pure antiestrogen ICI 182,780 has been shown to have antiprogestin activity in reporter gene constructs. Cell lines, naturally devoid of progesterone receptors (PR) were transfected with either the A or B forms of the human PR and a luciferase construct driven by a progesterone-response element (PRE). Because this system is an artificial one, our purpose was to determine whether these observations could be made in a human breast cancer cell line, naturally containing PR. We further evaluated the dose-response of ICI 182,780 and RU-486 (mifepristone) on PR and estrogen receptors (ER) in the presence of either progesterone, norgestrel or estradiol. These effects were measured using immunoassays for prostate-specific antigen (PSA) and human glandular kallikrein (hK2) and pS2. We found that ICI 182,780 blocked progesterone-stimulated PSA and hK2 production 100% at 10(-5) M, which decreased significantly by 10-6 M. This inhibition did not occur when norgestrel was the progestin used. RU-486 showed 100% blockade for both progestins at all concentrations used. We concluded that the antiprogestin activity of ICI 182,780 exists for progesterone only. This weak antiprogestin activity may be unlikely to have significant clinical implications.

Differential steroid hormone regulation of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) in breast cancer cell lines.

We have investigated the steroid hormone regulation of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) in the breast cancer cell lines BT-474, T-47D, MFM-223, MCF-7, ZR-75-1, MDA-MB-435, and BT-20. Using highly sensitive time-resolved fluorometric immunoassays, we were able to detect significant amounts of both kallikreins in tissue culture supernatants of BT-474, T-47D, and MFM-223 cells after hormonal stimulation. However, BT-474 cells produce much more hK2 than PSA, whereas the situation is reversed in T-47D cells. Furthermore, BT-474 cells produce, on absolute terms, about 500-1,000-fold more hK2 than T-47D cells. From all steroids tested, mibolerone, a synthetic non-metabolizable androgen, was the most potent stimulator for both kallikreins followed by the synthetic progestin norgestrel. Estradiol was able to induce production of small but significant amounts of hK2 and PSA in the BT-474 cell line, supporting the notion that there is a cross-talk between the estrogen and androgen hormone-receptor signaling pathways. MFM-223 is an androgen responsive cell line, devoid of other steroid hormone receptors, which is also capable of producing hK2 and PSA but at much lower amounts. MCF-7 and ZR-75-1 cell lines failed to produce any protein, even though they have similar steroid receptor content as the BT-474 and T-47D cell lines. This was also the case for MDA-MB-435, a cell line rich in androgen receptors. Our data suggest that the expression of the hK2 gene in breast cancer cell lines is mainly under the control of androgens and progestins, similarly to PSA. These cell lines may represent good models for studying the differential expression of these two genes and for identifying cellular factors (e.g. co-activators/co-repressors), which may modify the potency of expression after hormonal stimulation.

The normal epithelial cell-specific 1 (NES1) gene is up-regulated by steroid hormones in the breast carcinoma cell line BT-474.

The normal epithelial cell-specific 1 (NES1) gene encodes a serine protease which was found to be down-regulated in breast cancer. There is evidence that NES1 acts as a tumor suppressor gene in breast cancer cells. To further understand its role in breast tumorigenesis, we investigated the effect of estrogens, androgens, and progestins on NES1 gene expression, in the breast cancer cell line BT-474, at the transcription level. The reverse transcriptase polymerase chain reaction method was used to monitor changes in the NES1 mRNA. Our experiments showed that NES1 gene expression is up-regulated promptly in response to 17 beta-estradiol, 5 alpha-dihydrotestosterone (DHT) and norgestrel stimulation. NES1 gene mRNA started to increase 2 hours after estradiol stimulation and 8 hours after DHT stimulation. The stimulation of NES1 by estradiol can be dramatically blocked by the estrogen antagonists ICI 182,780 and 4-hydroxytamoxifen. Mifepristone (a synthetic antiprogestin) can partially block the up-regulation of the NES1 gene by norgestrel. Dose-response experiments indicated that the lowest stimulatory concentration of 17 beta-estradiol, DHT, and norgestrel is 10(-11) M, 10(-10) M, and 10(-10) M, respectively. The production of NES1 mRNA increased coordinately with increasing concentration of the stimulants. These results suggest that the NES1 gene is primarily regulated by estrogen, but also by androgen and progestin in the breast cancer cell line BT-474. It appears that NES1 may be involved in a pathway that counter balances the action of estrogens and androgens in steroid hormone responsive tissues.

Highly elevated levels of prostaglandin D synthase in the serum of patients with renal failure.

OBJECTIVES: To investigate whether prostaglandin D (PGD) synthase levels differ in the serum of patients with or without renal dysfunction. PGD synthase or beta-trace protein is a major constituent (approximately 3% of total protein) of human cerebrospinal fluid (CSF). We previously reported that PGD synthase levels in serum are approximately 40- to 60-fold lower than those in CSF. METHODS: We measured the PGD synthase concentration in various sera with a highly sensitive and specific immunofluorometric assay along with the serum creatinine level. Analysis for PGD synthase and creatinine was performed in 30 sera from non-renal failure subjects, in 7 sera from patients treated with continuous ambulatory peritoneal dialysis, and in 34 sera that were before and after hemodialysis samples from 17 patients with renal failure. RESULTS: Elevated creatinine concentration was observed in patients with renal insufficiency, as expected (Mann-Whitney P < 0.0001; chi-square P < 0.0001 ). We found that serum PGD synthase concentration from patients with renal failure is significantly elevated compared with the serum PGD synthase concentration from non-renal failure subjects (Mann-Whitney P < 0.0001; chi-square P < 0.0001). Approximately a 35-fold increase of serum PGD synthase is observed for patients with renal failure compared with non-renal failure subjects. Serum PGD synthase concentration is not affected by hemodialysis in acute renal failure patients (Mann-Whitney P = 0.918), unlike serum creatinine levels, which were decreased significantly after hemodialysis (Mann-Whitney P = 0.0001). CONCLUSIONS: We conclude that renal impairment is highly associated with elevated serum PGD synthase levels. Measurement of PGD synthase in serum is a new biochemical marker of renal insufficiency.

Modulation of androgen and progesterone receptors by phytochemicals in breast cancer cell lines.

We have used a tissue culture system based on breast carcinoma cell lines to investigate a large number of naturally occurring compounds and beverages for steroid hormone agonist and antagonist activity. The cell lines used, T-47D and BT-474, produce prostate specific antigen (PSA) upon stimulation with androgens, progestins, glucocorticoids and mineralocorticoids. This biomarker is secreted and can be measured in the tissue culture supernatant with very high sensitivity by an immunofluorometric procedure. Steroid hormone antagonist activity can be assessed with the same system by adding the candidate antagonist first and then stimulating the cells with a known agonist. By using this system we have identified three natural compounds, apigenin, naringenin and syringic acid which exhibited weak progestational activity and eleven other compounds which exhibited weak antiandrogenic/antiprogestational activity. Our study indicates that a significant number of natural compounds have the ability to bind to steroid hormone receptors and act as weak blockers. A fewer number of compounds not only bind to the receptors but they also mediate transcriptional activity, acting as agonists. The agonists and antagonists were active at levels around 10(-5) M, in accordance with previous reports for other phytochemicals. In comparison to synthetic and natural steroid hormones, the biological activity of these compounds is weaker by a factor of approximately 10(4)-fold.

Technical note. The serum concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine is increased in uremia.

Advanced glycation end products (AGEs) such as pentosidine and N epsilon-(carboxymethyl)lysine (CML) have been traditionally quantified by HPLC or gas chromatography--mass spectrometry (GC/MS). Enzyme-linked immunosorbent assays (ELISA) have been introduced as a convenient alternative to simplify the detection and measurement of AGEs in proteins and tissues, but some of these studies are limited by the lack of information on the structure of the epitopes recognized by antibodies to AGE-proteins. In this work we demonstrate that an antibody used in a previous study, reporting increased levels of AGEs in patients with diabetes or on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD), recognizes CML as its major epitope. We also show that there is a significant correlation between the concentration of AGEs in serum measured by ELISA and a GC/MS assay for CML in serum proteins. Both analyses yielded comparable results, with patients on CAPD and HD having about threefold higher AGE- or CML-concentrations in their serum. Our data suggest that ELISA assays for CML should be useful for the clinical measurement of AGEs in serum proteins.

Prostate-specific antigen in serum during the menstrual cycle.

We previously found that prostate-specific antigen (PSA) expression in the female breast is regulated by steroid hormones and their receptors. We have now examined whether the PSA concentration in serum changes during the menstrual cycle of healthy women. Among 14 women studied, 3 had serum PSA > or = 4 ng/L; their changes in PSA content during the menstrual cycle were studied in 7 informative cycles. We found that PSA concentrations in serum are highest during the mid- to late follicular phase, drop continuously with a half-life of 3-5 days between the late follicular phase and mid-cycle, and reach a minimum during the mid- to late luteal phase. PSA changes do not correlate with changes in lutropin (LH), follitropin (FSH), or estradiol concentrations. However, PSA peaks seem to follow the progesterone concentration peaks, with a delay of 10-12 days. Sera of some volunteers were tested for their ability to upregulate PSA protein and PSA mRNA in a tissue culture system based on the T-47D breast carcinoma cell line. Only sera obtained during the mid- to late luteal phase were able to upregulate the PSA mRNA and protein. In stimulation experiments in vitro, progesterone, but not LH, FSH, estradiol, human chorionic gonadotropin, prolactin, or growth hormone, was able to upregulate PSA mRNA and protein in the T-47D cell line. These data suggest that PSA is produced in a cyclical manner during the menstrual cycle.

Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer.

We have recently reported that about 30-40% of female breast tumours produce prostate-specific antigen (PSA) and that PSA production is associated with the presence of oestrogen (ER) and progesterone (PR) receptors. We have now developed a tissue culture system to study the regulation of the PSA gene in breast cancer. The breast carcinoma cell line T-47D produces PSA when stimulated by androgens, progestins and glucocorticoids/mineralocorticoids but not oestrogens. PSA mRNA appears approximately 2 h after stimulation; PSA protein appears after 4-8 h. Among 38 compounds tested, only androgens and progestins were able to stimulate PSA production at concentrations below 10(-9) M. Evidence that the progesterone and androgen receptors can regulate the PSA gene independently was provided as follows: (a) the progestin norgestimate, which does not bind to the androgen receptor, up-regulates the PSA gene at concentrations as low as 10(-10) M; (b) triamicinolone acetonide, which does not bind to the androgen receptor (AR) but binds to the PR, acts similarly to norgestimate; (c) the antiandrogen cyproterone acetate, which blocks the androgen receptor but has progestational activity, up-regulates the PSA gene at concentrations as low as 10(-10) M; (d) the antiprogestine mifepristone completely blocks the stimulation of the specific progestin norgestimate. Our tissue culture system identified androgen-progestin agonist activities of 17 alpha-ethinyloestradiol, the antioestrogen RU56, 187 and the antiprogestin mifepristone. Our data suggest that the expression of the PSA gene in the female breast is under the control of androgens and progestins. Our tissue culture system is a highly sensitive in vitro method for evaluating the biological activity of candidate compounds having agonist and antagonist steroid hormone activity.

Frequent detection of mutations in the 5' flanking region of the prostate-specific antigen gene in female breast cancer.

Prostate-specific antigen (PSA) is expressed in normal, hyperplastic and cancerous female breast tissue. Expression is regulated by steroid hormones. Some breast tumours produce very high levels of PSA, while other do not express any PSA. In this study, we selected three primary breast tumours which overexpressed PSA (PSA protein concentration in tumour cytosols > 4300 ng/l) and three tumours which were negative for PSA (< 1 ng/l). We extracted DNA and sequenced all five exons of the PSA gene. No mutations were found in the PSA coding sequence in any of the tumours. We identified only two polymorphic sites in exon 2. We also sequenced parts of the 5' flanking region of the PSA gene in five tumours. All tumour DNAs contained abnormalities which consisted of point mutations and deletions of 1-7 base pairs. Except for one tumour which had only a 3 base pair deletion, all other tumours had multiple abnormalities (up to seven in one tumour). The deletions occurred adjacent to direct repeats similarly to deletions seen in the p53 gene. Our data suggest that the coding sequence of PSA is not mutated in breast cancer. However, the 1.4 kb 5'-flanking region was mutated in all five tumours tested. The importance of this observation in relation to PSA gene regulation and breast cancer pathobiology remain to be determined.

Immunological quantification of advanced glycosylation end-products in the serum of patients on hemodialysis or CAPD.

We have developed an immunological procedure for measuring advanced glycosylation end-products (AGEs) in serum. Using this method, we measured AGEs in healthy volunteers, patients with diabetes, renal failure without treatment and in patients with renal failure, treated with hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). We found that AGEs were moderately elevated in diabetics without renal failure and highly elevated in CAPD and HD patients irrespective of their glycemic status. AGE levels correlated significantly with creatinine levels but not with levels of glucose or patient age or sex. AGE levels were reduced significantly post-hemodialysis. Preliminary experiments have shown that circulating AGEs have a molecular weight of approximately 1.5 to 2.0 kDa. More studies are needed to establish if AGE measurements in serum are prognostic indicators of the complications of either diabetes or renal failure.

Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines.

We demonstrate that the steroid hormone receptor-positive breast carcinoma cell lines T-47D and MCF-7 can be induced by androgens, progestins, mineralocorticosteroids, glucocorticosteroids, and antiestrogens, to produce prostate specific antigen (PSA). Estrogens failed to induce such stimulation in both cell lines and, in addition, were able to block the induction by androgens in the cell line T-47D. These data support and extend our previous report on PSA production by breast tumors and describe an in vitro system which can be used to study the phenomenon for possible application in prognosis and design of new therapy.

Urine citrate and renal stone disease.

Calcium stone disease is attributable to supersaturation of the urine with calcium and other salts, the presence of substances that promote crystallization and a deficiency of inhibitors of crystallization. Citrate is a potent inhibitor of calcium oxalate and calcium phosphate stone formation whose excretion is diminished in some patients with stone disease owing to idiopathic causes or secondary factors such as bowel disease and use of thiazides. The pH within the proximal tubule cells is an important determinant of citrate excretion. Multivariate analysis has shown that the urine concentrations of calcium and citrate are the most important factors in stone formation. In uncontrolled studies potassium citrate, which increases urinary citrate excretion, appears to be promising as a therapeutic agent for patients with stone disease and hypocitraturia refractory to other treatment. On the other hand, there are potential drawbacks to sodium alkali therapy, such as the precipitation of calcium phosphates.

Control of hyperoxaluria with large doses of pyridoxine in patients with kidney stones.

Pyridoxine in doses of 250-500 mg daily by mouth was administered to 12 patients suffering from recurrent calcium oxalate renal calculi and idiopathic hyperoxaluria. This therapy decreased urinary oxalate excretion significantly (p less than 0.025) during up to 18 months of treatment. In that period eight patients showed no evidence of active stone disease; three showed slight increase in the size of their old stone(s) and one patient formed one new stone. None of these patients developed any significant complications of the therapy. These findings support the view that pyridoxine in pharmacological doses is useful in the control of elevated urinary oxalate excretion in patients with recurrent renal oxalate calculi.