One year of surgical mask testing at the University of Bologna labs: Lessons learned from data analysis.

The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%.

Genome-wide paternal uniparental disomy as a cause of Beckwith-Wiedemann syndrome associated with recurrent virilizing adrenocortical tumors.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by fetal macrosomia, macroglossia, and abdominal wall defects. BWS patients are at risk to develop Wilms tumor, neuroblastoma, hepatoblastoma, and adrenal tumors. A young woman with BWS features, but with inconclusive genetic evidence for the disease, came to clinical observation for signs of virilization at the age of 16 years. An adrenocortical tumor was diagnosed and surgically resected. The tumor underwent 2 local relapses that were also surgically treated. The patient was also operated to remove a breast fibroadenoma. SNP arrays were used to analyze chromosome abnormalities in normal and tumor samples from the patient and her parents. The patient presented genome-wide mosaic paternal uniparental disomy (patUPD) both in the adrenocortical and the breast tumors, with different degrees of loss of heterozygosity (LOH). The more recent relapses of the adrenocortical tumor showed a loss of part of chromosome 17p that was absent in the first tumor. Analysis of a skin biopsy sample also showed mosaic patUPD with partial LOH, while no LOH was detected in leukocyte DNA. This case shows that virilizing adrenocortical tumors may be a clinical feature of patients with BWS. The SNP array technology is useful to diagnose genome-wide patUPD mosaicism in BWS patients with an inconclusive molecular diagnosis and underlines the tumorigenic potential of the absence of the maternal genome combined with an excess of the paternal genome.

Genomic analysis of sexual dimorphism of gene expression in the mouse adrenal gland.

A relevant gender difference exists in adrenal physiology and propensity to disease. In mice, a remarkable sexual dimorphism is present in several components of the hypothalamic-pituitary-adrenal axis, with females displaying higher adrenal weight, plasma ACTH, corticosterone, and aldosterone levels than males. The molecular bases of this sexual dimorphism are little known. We have compared global gene expression profiles in males vs. female mouse adrenal glands and also studied the effect that testosterone treatment and castration have on adrenal gene expression in female vs. male mice, respectively. Our study evidenced a set of 71 genes that are coordinately modulated according to sex and hormonal treatments and represent the core sexually dimorphic expression program in the mouse adrenal gland. Moreover, we show that some genes involved in steroid metabolism have a remarkable sexual dimorphic expression and identify new potential markers for the adrenal X-zone, a transitory cellular layer in the inner adrenal cortex, which spontaneously regresses at puberty in males and during the first pregnancy in females and has an uncertain physiological role. Finally, sexually dimorphic expression of the transcriptional regulators Nr5a1 and Nr0b1 may explain at least in part the differences in adrenal steroidogenesis between sexes.

Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA.

The DAX-1 (NR0B1) gene encodes an unusual member of the nuclear hormone receptor superfamily which acts as a transcriptional repressor. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG). We have studied the intracellular localization of the DAX-1 protein in human adrenal cortex and mouse Leydig tumor cells and found it to be both nuclear and cytoplasmic. A significant proportion of DAX-1 is associated with polyribosomes and is found complexed with polyadenylated RNA. DAX-1 directly binds to RNA, two domains within the protein being responsible for cooperative binding activity and specificity. Mutations in DAX-1 found in AHC-HHG patients significantly impair RNA binding. These findings reveal that DAX-1 plays multiple regulatory roles at the transcriptional and posttranscriptional levels.

Hormonal and developmental regulation of DAX-1 expression in Sertoli cells.

Mutations in the human DAX-1 gene lead to X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. DAX-1 has been proposed to play a role in steroidogenesis because it is highly expressed in adrenocortical and testicular Leydig cells and because loss-of-function mutations lead to low serum levels of steroid hormones. Recent reports of DAX-1 expression in hypothalamus and pituitary, however, suggest additional functions for this protein. Here we demonstrate that DAX-1 is expressed in Sertoli cells of rat testis. This expression is regulated during spermatogenesis and peaks during the androgen-sensitive phase of the spermatogenic cycle. In addition, we show that DAX-1 expression in Sertoli cells is regulated developmentally. Maximum levels are present in the rat between postnatal days 20 and 30, during the first spermatogenic wave. Moreover, we show that activation of the cAMP-signaling pathway by the pituitary hormone FSH leads to a potent down-regulation of DAX-1 expression in cultured Sertoli cells. This down-regulation requires transcription and de novo protein synthesis. Taken together, these data indicate that DAX-1 expression in Sertoli cells may influence the development of spermatogenic cells in response to steroid and pituitary hormones.

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Heat shock interferes with steroidogenesis by reducing transcription of the steroidogenic acute regulatory protein gene.

A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.

A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita.

The DAX-1 gene encodes an unusual member of the nuclear hormone receptor superfamily. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. We have shown that DAX-1 binds to hairpin secondary structures and blocks steroidogenesis in adrenal cells via transcriptional repression of the steroidogenic acute regulatory protein (StAR) promoter. Here we have investigated the molecular mechanism of DAX-1-mediated repression. We show that the DAX-1 C terminus contains a potent transcriptional silencing activity, which can be transferred to a heterologous DNA-binding domain. Deletion analysis and modeling of DAX-1 structure identify two cooperating domains required for the silencing function, one located within helix H3 and the other within H12. The silencing function is cell- and promoter-specific. Strikingly, two point mutations (R267P and deltaV269) found in adrenal hypoplasia patients impair silencing. These findings suggest that transcriptional silencing by DAX-1 plays a critical role in the pathogenesis of adrenal hypoplasia congenita.

Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development.

Mutations of the orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, cause complex endocrine phenotypes that include impaired adrenal development and hypogonadotrophic hypogonadism. These similar phenotypes suggest that SF-1 and DAX-1 act in the same pathway(s) of endocrine development. To explore this model, we now compare directly their sites of expression. In mouse embryos, SF-1 expression in the urogenital ridge and brain either preceded or coincided with Dax-1 expression, with coordinate expression thereafter in the adrenal cortex, testis, ovary, hypothalamus, and anterior pituitary. The striking colocalization of SF-1 and Dax-1 supports the model that they are intimately linked in a common pathway of endocrine development. The slightly earlier onset of SF-1 expression and its ability to bind specifically to a conserved sequence in the Dax-1 5'-flanking region suggested that SF-1 may activate Dax-1 expression. However, promoter activity of Dax-1 5'-flanking sequences did not require this potential SF-1-responsive element, and Dax-1 expression was unimpaired in knockout mice lacking SF-1, establishing that SF-1 is not required for Dax-1 gene expression in these settings. Although the precise mechanisms remain to be established and may be multifactorial, our results strongly suggest that these two orphan nuclear receptors interact in a common pathway of endocrine development.

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3beta-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3beta-HSD promoters. An inappropriate steroidogenic block in the male fetus might have an important role in the pathogenesis of sex reversal syndromes caused by a duplication of the genomic region of the X chromosome containing the DAX-1 gene.

DAX-1 expression in human adrenocortical neoplasms: implications for steroidogenesis.

The DAX-1 gene encodes an orphan nuclear hormone receptor essential for normal fetal development of the adrenal cortex. Recently, DAX-1 has been shown to act as a transcriptional repressor of steroidogenic acute regulatory protein gene expression (StAR), suppressing steroidogenesis. We, therefore, investigated the expression of DAX-1 in a variety of adrenocortical tumors and compared the results with StAR mRNA expression. We found low or absent DAX-1 expression in aldosterone-producing adenomas (n = 11: 35 +/- 11%; normal adrenals: 100 +/- 17%) and in aldosterone-producing adrenocortical carcinomas (n = 2: 24 and 36%). Cortisol-producing adenomas showed intermediate DAX-1 expression (n = 8; 92 +/- 16), as did 3 non-aldosterone-producing carcinomas (72, 132 and 132%). High DAX-1 expression was present in nonfunctional adenomas (n = 3; 160 +/- 17%). In contrast to DAX-1, StAR mRNA expression did not show significant variations between groups. We did not detect the expected negative correlation between DAX-1 and StAR in adrenocortical tumors. These data suggest that high DAX-1 expression in adrenocortical tumors is associated with a non-functional phenotype whereas low DAX-1 expression favors mineralocorticoid secretion. These effects on steroidogenesis are mediated by mechanisms other than repression of StAR gene expression. Our results indicate that DAX-1 may be one of the factors influencing the steroid biosynthesis of adrenocortical neoplasms.

Recombinant growth hormone (GH) therapy in GH-deficient adults: a long-term controlled study on daily versus thrice weekly injections.

Currently, replacement recombinant GH (rGH) therapy in GH-deficient (GHD) adults is performed in daily injections. This modality of treatment is not complied with by the totality of GHD patients, who are supposed to receive life-long replacement. The aim of our study was to compare daily vs. thrice weekly (TIW) rGH injection effects on lipid profile, body composition, bone metabolism, and bone density in 34 GHD patients (13 women and 21 men; median age, 39 yr; range, 30-55 yr) randomly assigned to different therapeutic regimens. Group A included 18 patients receiving daily rGH injections, and group B included 16 patients receiving TIW injections of rGH. The starting dose of rGH was 10 microg/kg x day in both groups. Subsequently, the dose was adjusted to maintain serum insulin-like growth factor I (IGF-I) concentrations in the normal age-adjusted range. IGF-I levels were assessed before and after 1, 3, 6, and 12 months of rGH treatment, and lipid profile, body composition, bone metabolism, and bone density were evaluated before and after 6 and 12 months of treatment. Thirty-four healthy subjects served as controls. In the basal condition, lipid profile, body composition, bone metabolism, and bone density were significantly different in patients compared to controls. Conversely, patients included in groups A and B had similar serum IGF-I levels, lipid profile, body composition, bone metabolism, and bone density. After 3 months of rGH treatment, IGF-I levels were normalized in 15 of 18 patients (83.3%) in group A and in 7 of 16 patients (43.7%) in group B (chi2 = 4.21; P = 0.04). At this time point, serum IGF-I levels in patients in group A (202+/-57.5 microg/L) were significantly higher than those in patients in group B (155+/-45.1 microg/L; P = 0.001). After 6 months of therapy, serum IGF-I levels were normalized in all patients and were similar in both groups (223+/-35.2 vs. 212+/-41.4 microg/L, A vs. B, respectively). IGF-I levels remained normal until the 12-month follow-up. After 6 months of rGH replacement, total cholesterol, low density lipoprotein cholesterol, triglycerides, bioelectrical impedance, and body fat mass were significantly reduced, whereas high density lipoprotein cholesterol levels and lean body mass were significantly increased in both groups of patients, without any difference between them. No further change in lipid profile and body composition was observed after 12 months of treatment. Serum bone GLA protein and procollagen III levels were significantly increased after 6 months, and a downward trend was observed after 12 months of rGH replacement. However, a slight, but significant, increase in bone mineral density was observed in both groups only after 12 months (P = 0.0001). All patients in group B had good compliance to the TIW treatment, whereas 5 patients in group A had poor compliance to the treatment (chi2 = 3.2; P = 0.07). In conclusion, our randomized, prospective, and controlled study confirmed that rGH therapy with TIW injection regimen is effective in normalizing IGF-I levels and improving lipid profile, body composition, bone metabolism, and bone density. It also demonstrated that this efficacy is comparable to that observed in patients treated with daily rhGH therapy, with few side-effects and good compliance.

High serum osteoprotegerin levels in patients with hyperthyroidism: effect of medical treatment.

This study was aimed at evaluating serum osteoprotegerin (OPG) concentrations in a cohort of patients with hyperthyroidism before and after methimazole (MMI) treatment. One hundred fourteen hyperthyroid patients [93 with Graves disease (GD) and 21 with toxic nodular goitre (TNG)] and 68 matched for sex and age healthy subjects were evaluated for serum free-thyroxine (FT4), free-triiodiothyronine (FT3), thyrotropin (TSH), TSH receptor antibodies (TRAb), bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), OPG levels, and bone mineral density (BMD). In hyperthyroid patients, the biochemical evaluations were performed before and after 6 and 12 months of MMI treatment, whereas BMD was measured at baseline and after 12 months of treatment. Hyperthyroidism was more severe in GD than TNG patients. Serum OPG levels were found to be significantly higher in hyperthyroid patients than in the healthy subjects (4.3 pmol/l, range: 1.6-12.0, vs. 2.2 pmol/l, range: 1.4-6.0; P < 0.001), the values being higher in GD patients than TNG. A significant correlation between serum OPG levels and age was found in the healthy subjects (r: 0.48; P < 0.001) but not in hyperthyroid patients (r: -0.03; P = 0.8). In the healthy subjects, serum OPG levels were also positively correlated with both serum FT4 (r: 0.23; P = 0.03) and FT3 (r: 0.24; P = 0.04) levels. In hyperthyroid patients, however, serum OPG was still correlated with FT3 levels (r: 0.38; P < 0.001), whereas the correlation with serum FT4 was lost (r: 0.19; P = 0.06). In hyperthyroid patients, but not in the healthy subjects, serum OPG levels were correlated positively with CrossLaps (r: 0.20; P = 0.03) and negatively with BALP (r: -0.24; P = 0.01) and BMD (r: -0.33; P = 0.01). After 6 months of MMI treatment, serum OPG concentrations decreased significantly in TNG patients (from 3.5 pmol/l, range: 1.6-8.0, to 2.3 pmol/l, range: 1.0-4.3; P < 0.001), whereas a not significant change in OPG levels occurred in GD patients (from 4.8 pmol/l, range: 1.8-12.0, to 4.2 pmol/l, range: 1.0-14.0; P = 0.7). At Month 12 of treatment, serum OPG concentrations were significantly lower than those measured at baseline in both TNG (2.5 pmol/l, range: 1.0-3.1, vs. 3.5 pmol/l, range: 1.6-8.0; P < 0.001) and GD (2.1 pmol/l, range: 1.0-8.6, vs. 4.8 pmol/l, range: 1.8-12.0; P < 0.001). At this time, no significant differences in serum OPG, CrossLaps, and BALP values were found between patients and control subjects. At the end of follow-up, BMD was higher than those measured at baseline but still significantly lower than those measured in the control subjects. This study shows that hyperthyroid patients have serum OPG concentrations significantly higher in comparison with euthyroid subjects, in relation to thyroid hormone excess and high bone turnover. Medical treatment of hyperthyroidism normalizes serum OPG levels in temporal relationship with the normalization of bone metabolism markers, even in presence of persistent abnormal bone structure as determined by ultrasonography.

Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1.

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.

Commercial serum-free media: hybridoma growth and monoclonal antibody production.

Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media. However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used. In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines. Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA). Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions. Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines. We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.

Serum amyloid A protein concentration in bone marrow transplantation for beta thalassaemia.

AIMS: To investigate whether serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations could be used in the management of beta thalassaemic patients undergoing bone marrow transplantation (BMT). METHODS: Serum SAA and CRP concentrations were determined in paired samples from 66 patients with beta thalassaemia before and after BMT. Serum SAA concentrations were determined by an enzyme linked immunoassay (EIA); serum CRP concentrations were determined by a nephelometric assay. RESULTS: Serum SAA concentrations before transplantation were significantly higher in the group that subsequently rejected the transplant than the group without complications. SAA concentrations increased after BMT in acute graft versus host disease (GvHD) and rejection. No significant increase in SAA or CRP was found in chronic GvHD. Increases in serum in SAA and CRP concentrations were not related to concomitant infection episodes. CONCLUSIONS: The different acute phase response in acute GvHD and rejection compared with chronic GvHD suggests that different immunopathogenic mechanisms are responsible.

Increased serum concentrations of tumour necrosis factor in beta thalassaemia: effect of bone marrow transplantation.

AIMS: Serum concentrations of tumour necrosis factor-alpha (TNF) were determined in beta thalassemic patients before and after bone marrow transplantation (BMT) to evaluate whether changes in TNF concentrations after BMT were related to immune mediated complications. METHODS: Serum TNF concentrations were determined by enzyme linked immunoassay (EIA) in paired samples from 71 patients with beta thalassemia before and after BMT. Serial samples from 13 patients were also studied for up to six months after BMT. Forty one normal healthy children matched for sex and age were studied as controls. RESULTS: beta thalassemic patients had high serum TNF concentrations before transplantation compared with controls. These were not related to sex, age, duration of disease, number of blood transfusions, transferrin concentrations or splenectomy. DQw1 positive patients showed significantly lower TNF concentrations than non-DQw1 cases. Patients with severe liver fibrosis had significantly higher TNF concentrations. No correlation was found between TNF values and BMT outcome before transplantation but TNF alpha values fell significantly after BMT. The decrease persisted only in patients with successful engraftment. In serial samples studied for up to six months after BMT, TNF values decreased but in four out of five patients with graft rejection and in all five with acute graft versus host disease (GVHD) sharp increases occurred at the time of clinical symptoms. No correlation was found between the degree of GVHD and serum TNF-alpha concentrations nor between TNF-alpha concentrations after BMT and the presence of bacterial, viral, and fungal infections. CONCLUSIONS: About 50% of beta thalassemic patients have increased serum TNF, and the changes after BMT are related to the occurrence of immune mediate complications. The persistence of low TNF concentrations after successful engraftment may be due to the preparative regimen and the lack of adverse immune reactions.

Ly-6A/E gene is widely expressed among transformed nonhematopoietic cells. Autocrine modulation by interferon.

Expression of the Ly-6A/E gene by transformed cells was investigated in 14 cell cultures of C57BL/6 and BALB/c mouse origin derived from spontaneous or chemically-induced non-hematopoietic tumours and from cells transformed by SV40 virus. Histologic types included carcinomas, sarcomas and a melanoma. Thirteen out of 14 cell cultures expressed membrane Ly-6A/E antigens; only the B16-A melanoma was negative, but it expressed Ly-6A/E after incubation with IFN-gamma. The effect of in vitro permanence was studied on early (< 10) and late (> 20) passages of B16-A melanoma and MN/MCA1 fibrosarcoma. Late passage B16-A cells were Ly-6A/E-negative and refractory to induction of Ly-6A/E (but not of H-2 antigens) by IFN-alpha, IFN-beta, or IFN-gamma; MN/MCA1 maintained a high expression of Ly-6A/E, but no increase was induced by IFNs. It was found that both early and late in vitro passages of MN/MCA1 actively produced IFN-alpha/beta. The analysis of cells of fibroblastic origin revealed a significant correlation between IFN release in the culture medium and Ly-6A/E levels. Culture of fibrosarcoma cells in the presence of an anti-IFN-alpha/beta serum reduced Ly-6A/E expression, thus indicating the existence of an autocrine loop.

Synergism between gamma interferon and doxorubicin in a human MDR colon adenocarcinoma cell line.

Recent interest in cancer therapy derives from the ability of interferons to synergistically increase the activity of chemotherapeutic agents. To understand the biological basis of this synergism we evaluated the effects of human recombinant IFN-gamma on the expression of the mdr1 gene and on the cellular growth of a human colon adenocarcinoma cell line (LoVo) and its MDR subline (LoVo/Dx) after coincubation with doxorubicin. Treatment with IFN-gamma showed unchanged levels of MDR1-glycoprotein, no perturbation on cell cycle distribution and a significant reduction of colony formation in both lines (P < 0.05) starting from 100 U/ml. A synergistic effect was observed in the LoVo/Dx cell line when doxorubicin was added after exposure to 0.1-10 U/ml of IFN-gamma. Our data indicate that the effects of IFN-gamma, independent from action on cell proliferation and from modulation of p-glycoprotein expression, are a cause of the synergistic activity between this lymphokine and conventional chemotherapeutic agents such as doxorubicin.

The momentum of human behavior in a natural setting.

Adults with mental retardation in a group home received popcorn or coffee reinforcers for sorting plastic dinnerware. In Part 1 of the experiment, reinforcers were dispensed according to a variable-interval 60-s schedule for sorting dinnerware of one color and according to a variable-interval 240-s schedule for sorting dinnerware of a different color in successive components of a multiple schedule. Sorting rates were similar in baseline, but when a video program was shown concurrently, sorting of dinnerware was more resistant to distraction when correlated with a higher rate of reinforcement. In Part 2 of the experiment, popcorn or coffee reinforcers were contingent upon sorting both colors of dinnerware according to variable-interval 60-s schedules, but additional reinforcers were given independently of sorting according to a variable-time 30-s schedule during one dinnerware-color component. Baseline sorting rate was lower but resistance to distraction by the video program was greater in the component with additional variable-time reinforcers. These results demonstrate that resistance to distraction depends on the rate of reinforcers obtained in the presence of component stimuli but is independent of baseline response rates and response-reinforcer contingencies. Moreover, these results are similar to those obtained in laboratory studies with pigeons, demonstrating that the determination of resistance to change by stimulus-reinforcer relations is not confined to controlled laboratory settings or unique to the pigeon.