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.

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.

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.

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.