REGOMA-OSS: a large, Italian, multicenter, prospective, observational study evaluating the efficacy and safety of regorafenib in patients with recurrent glioblastoma.

BACKGROUND: In the randomized phase II REGOMA trial, regorafenib showed promising activity in patients with recurrent glioblastoma. We conducted a large, multicenter, prospective, observational study to confirm the REGOMA data in a real-world setting. PATIENTS AND METHODS: The major inclusion criteria were histologically confirmed diagnosis of glioblastoma according to the World Health Organization (WHO) 2016 classification and relapse after radiotherapy with concurrent/adjuvant temozolomide treatment, good performance status [Eastern Cooperative Oncology Group performance status (ECOG PS 0-1)] and good liver function. Regorafenib was administered at the standard dose of 160 mg/day for 3 weeks on/1 week off. Brain magnetic resonance imaging was carried out within 14 days before starting regorafenib and every 8-12 weeks. The primary endpoint was overall survival (OS). The secondary endpoints were progression-free survival (PFS), objective response rate, disease control rate (DCR), safety and health-related quality of life. The Response Assessment in Neuro-Oncology (RANO) criteria were used for response evaluation and Common Terminology Criteria for Adverse Events (CTCAE) version 5 for assessment of adverse events (AEs). RESULTS: From September 2020 to October 2022, 190 patients with recurrent glioblastoma were enrolled from 30 cancer centers in Italy: their median age was 58.5 years [interquartile range (IQR) 53-67 years], 68% were male and 85 (44.7%) were in optimal clinical condition (ECOG PS 0). The number of patients taking steroids at baseline was 113 (60%); the second surgery was carried out in 39 (20.5%). O6-methylguanine-DNA methyltransferase (MGMT) was methylated in 80 patients (50.3%) and 147 (92.4%) of the patients analyzed had isocitrate dehydrogenase (IDH) wild type. The median follow-up period was 20 months (IQR 15.6-25.5 months). The median OS was 7.9 months ([95% confidence interval (CI) 6.5-9.2 months] and the median PFS was 2.6 months (95% CI 2.3-2.9 months). Radiological response was partial response and stable disease in 13 (7.3%) and 26 (14.6%) patients, respectively, with a DCR of 21.9%. The median number of regorafenib cycles per patient was 3 (IQR 2.0-4.0). Grade 3-4 drug-related adverse events were reported in 22.6% of patients. A dose reduction due to AEs was required in 36% of patients. No deaths were considered as treatment-related AEs. CONCLUSIONS: This large, real-world observational study showed similar OS with better tolerability of regorafenib in patients with relapsed glioblastoma compared with the REGOMA study.

Influence of melatonin receptor 1A gene polymorphisms on seasonal reproduction in Sarda ewes with different body condition scores and ages.

In several species, circadian changes in melatonin concentrations play a key role in the photoperiodic control of seasonality. In sheep, two silent mutations in the melatonin receptor 1A gene (MTNR1A) at positions 606 and 612 of the exon II are associated with seasonal reproduction. However, in some sheep breeds, no relationships have been found between MTNR1A polymorphisms and reproductive seasonality. This lack of relationship could be due to effects of breed, body condition, age, and/or environmental conditions. Thus, the present study was conducted with the Sarda sheep breed with the aim of documenting the effect of MTNR1A gene polymorphisms on reproductive resumption and to evaluate whether such this effect was modified by differences in body condition score (BCS) and age. Six hundred three- to six-year-old multiparous ewes with BCSs between 2.5 and 3.5 were selected. Genomic DNA was extracted and subjected to PCR to amplify the ovine exon II of the MTNR1A gene. The amplicons were subjected to digestion with the restriction enzymes RsaI and MnlI to detect the T606C and A612G polymorphisms, respectively. Ewes carrying the G/G, G/A, C/C, and C/T genotypes exhibited higher fertility rates (P<0.05) and fewer numbers of days between the introduction of rams and parturition (P<0.05) than did the A/A and T/T genotypes. The data revealed that the MTNR1A gene polymorphisms influenced spring reproductive resumption in the Sarda sheep breed. Moreover, the data also indicated that, over the limited ranges evaluated in this study, BCS and age had no significant influence on reproductive activity.

Effect of genotype at the MTNR1A locus and melatonin treatment on first conception in Sarda ewe lambs.

A sample of 423 Sarda ewe lambs from three different farms was used to evaluate the effect of one or two melatonin implants on the time of first conception. On each farm, 141 animals were divided into three groups. On June 30 these animals received either no treatment (Group C), 18 mg melatonin (Group M1, one implant), or 18 + 18 mg melatonin (Group M2, two implants). Thirty-five days after treatment, rams were introduced in the ewe lambs flock and subsequently removed after 40 days. Lambing dates were recorded between 150 and 190 days from the first day of male introduction. Genotyping and sequencing of the MT1 exon 2 were carried out to analyze the structure and the possible influence of the MT1 receptor gene on reproductive response to melatonin treatment. Melatonin-treated animals had a higher rate of pregnancy (P < 0.05) and lambed earlier (P < 0.05) compared with untreated animals. Single nucleotide polymorphisms were found in exon II of MT1 gene at positions C606T and G612A leading to genotypes C/C, C/T or T/T and +/+, +/- and -/-, respectively. Melatonin-treated animals of +/+ genotype showed a higher number of pregnancies (P < 0.05) and lambed earlier (P < 0.05) compared to untreated animals of the same genotype. Melatonin treatment did not affect reproductive activity in any other genotype analyzed. No correlation between genotype and the time of first conception was found in untreated animals. Concluding data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that +/+ genotype is able to influence reproductive response to melatonin treatment.

Changes in nutritional status and body composition during enzyme replacement therapy in adult-onset type II glycogenosis.

BACKGROUND: In adult glycogen storage disease type II (GSDII), a single-gene mutation causes reduction of the lysosomal enzyme acid alpha-glucosidse. This produces a chronic proximal myopathy with respiratory involvement. Enzyme replacement treatment (ERT) has recently become available and is expected to improve muscle strength. This should result in increased lean body mass. In this study we evaluate body composition and nutritional status in GSDII, and assess whether these parameters changed during treatment. METHODS: Seventeen patients with late-onset GSDII, aged 52.6 +/- 16.8 years, received ERT for >18 months. Dietary habits and metabolic profiles of glucids, lipids, and proteins were assessed. Body composition was calculated using anthropometry and bioelectrical impedence analysis. RESULTS: On inclusion, we found increased fat mass (FM) in five patients in severe disease stage; all had normal body mass index (BMI). FM correlated inversely, and lean mass (LM) directly, with creatine kinase, prealbumin and albumin levels. After treatment, BMI and FM significantly increased, while LM only showed a trend toward increase. Prealbumin and albumin levels increased as early as after the first months of ERT. DISCUSSION: Body mass index value may underestimate FM in patients in severe stage of disease, due to altered body composition. In severely affected patients, laboratory parameters revealed a relative protein malnutrition, that was reversed by ERT, this reflecting restoration of normal muscle metabolic pathways. Increased BMI may indicate a reduction in energy consumption during exercise or respiration, along with clinical improvement.

Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed.

The aim was to study the polymorphisms of the melatonin receptor 1A gene (MTNR1A) and its relationship with seasonal reproduction in the Sarda sheep breed. Four-thousand multiparous ewes reared under natural photoperiod were randomly chosen. Genomic DNA was extracted and subjected to PCR for the amplification of the main part of exon II of the ovine MTNR1A gene (GenBank U14109). PCR products were subjected to restriction enzymes MnlI and RsaI and placed into +/+, +/- or -/- group for MnlI and C/C, C/T or T/T group for RsaI. Samples were cloned and sequenced. The sequences were aligned with the U14109 sequence of GenBank. Data were subjected to allelic frequency analysis and to the chi(2) test in order to evaluate the link between genotype and reproductive activity. After MnlI digestion, allelic frequency was 0.78 for allele +and 0.22 for allele -; genotype frequency of the +/+ homozygote was 68%, 20.5% for +/- and 11.5% for -/-. After RsaI, allelic frequency was 0.66 for allele C and 0.34 for allele T; genotype frequency of the C/C homozygote was 53.5%, 26% for C/T and 20.5% for T/T. The population was in Hardy-Weinberg disequilibrium both for the MnlI and RsaI. Lambing frequency of +/+ genotype ewes was higher in the period September-December while for -/- genotype in January-April (P<0.01). Lambing of C/C genotype ewes showed a higher frequency in September-December while for T/T genotype in January-April (P<0.01). Results confirmed that the polymorphism of the MTNR1A locus was also present in the Sarda with a higher incidence of the +/+ and C/C genotypes. The animals that carried one of these two gene isoforms showed a not seasonal reproductive activity with the lambing period in September-December.

Relationship between MTNR1A melatonin receptor gene polymorphism and seasonal reproduction in different goat breeds.

The reproductive activity of goats bred in temperate latitude follows a seasonal pattern, influenced by annual variation in day length. Daily variation in pineal melatonin secretion is the neuroendocrine signal recognized by animals through the link between this hormone and melatonin receptor 1a (MTNR1A). A total of 345 goats of different breeds (225 Sarda, 30 Saanen, 30 Chamois Coloured, 30 Maltese and 30 Nubian) with a kidding period in October-December or January-March were analysed to verify if a link exists between the structure of the receptor gene and reproductive activity. The main part of exon II of MTNR1A gene was amplified by PCR and then digested with MnlI and RsaI to prove the presence of restriction sites. Sequencing of 20 cloned samples and 20 purified samples permitted comparison with previously published sequences. No polymorphism was found using MnlI enzyme, as all 345 samples showed the cleavage site in position 605 and all the goats were MM genotype. However, using RsaI enzyme, some Sarda goats, showed a polymorphic site in position 53. Nine Sarda goats were R/r genotype, lacking this cleavage site only in one allele, while the other animals, both Sarda and the other breeds, presented the cleavage site in both the alleles and were thus R/R genotype. No r/r genotype was found in any of the breeds. In Sarda goats the allelic frequency was 0.98 for R allele and 0.02 for r allele; genotypic frequency was 96.00% for R/R genotype and 4.00% for R/r genotype. A strong link emerged from statistical analysis (P<0.001) between R/r genotype and reproductive activity, which was strongly influenced by photoperiod. Sequencing indicated six nucleotide changes that did not induce any amino acid change. Data showed that polymorphism was present and that it influences reproductive activity only in the Sarda breed.

The effect of shearing procedures on blood levels of growth hormone, cortisol and other stress haematochemical parameters in Sarda sheep.

The aim of this research was to investigate how growth hormone (GH) cortisol and some haematochemical parameters could be modified by the stress caused by the stages of shearing in Sarda breed sheep. Five groups of 10 sheep each were formed. Group A, only separated from the flock; Group B, only tied; Group C, both tied and shorn (animals in these three groups were ewe lambs shorn for the first time); Group D, adult females both tied and shorn; and Group E, adult entire males both tied and shorn (animals in these two groups had been shorn previously). Five blood samples were taken from each animal: the day before treatment (first sample); at the start of the treatment (second sample); in the middle of shearing for Groups C, D and E, 10 min after separation in Group A and 10 min after tying in Group B (third sample); at the end of treatment (fourth sample); and on the day after treatment (fifth sample). Plasma GH levels showed a decrease (P < 0.01) in Groups A, B, C and D during treatment (third and fourth samples), while Group E only at the end of shearing (fourth sample). In the third sample, the highest GH levels were recorded for Group E (P < 0.05), while it was recorded in the fourth sample for Groups A and E (P < 0.05). Cortisol levels showed a clear increase (P < 0.01) in all groups during treatment, but Group A showed a decrease in the fourth sample in comparison to the third sample. Males in the second, third and fourth sample and Group A only in the fourth sample showed lower cortisol levels when compared with the other groups (P < 0.05). Plasma glucose levels showed an increase (P < 0.01) in all groups during treatment but Groups B, C and E showed the highest values (P < 0.05). Magnesium (Mg) showed an increase in all groups in the third and fourth sample, while sodium (Na), in the same samples, only in Groups B, C and D. Potassium (K) values showed a significant decrease (P < 0.05) only in Groups C and D at the end of shearing. These results show that GH secretion is influenced by all the stress procedure: separation, tying and shearing. Shearing, even if necessary for animals, causes a significant change of the blood parameters involved in the stress response.

[A case of Caffey-De Toni-Silvermann disease with mandibular localization]. / Un caso di malattia di Caffey-De Toni-Silverman a localizzazione mandibolare.

Report of a case of infantile cortical hyperostosis with localitation in the jaw-bone in a female child five month old. Spontaneous resolution, not relapse. There weren't other affected subjects in the same family. Unknown etiology. Discussion about the differential diagnosis and the therapeutic ways described in literature.

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death.

An optimal stimulation of CD4+ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells (APC). The intercellular adhesion molecule-1 (ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin (IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones (TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as Th0 (IL-4 plus interferon-gamma) or Th2 (IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4+ and CD8+ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones (with Th0- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibody response to the Ro/La particle may predict outcome in neonatal lupus erythematosus.

This study was undertaken to determine the role of antibodies against both recombinant Ro (r-Ro) and La (r-La) proteins and polypeptides derived from the recombinant La protein in predicting fetal and neonatal outcome in children at risk to develop neonatal lupus erythematosus (NLE). All sera were obtained in the perinatal period and quantitative ELISA assays were used. We collected 41 maternal sera within 2 months of delivery of a child with NLE (21 with congenital heart disease block (CHB) and 20 with dermatologic NLE) and 19 sera from anti-Ro and/or anti-La antibody-positive mothers with systemic lupus erythematosus (SLE) who delivered a child without NLE. All sera were tested for anti-r-La and anti-r-Ro antibodies by ELISA, and most sera were tested for antibodies directed against La polypeptides by immunoblot. We found significantly higher anti-r-La antibody levels in the sera from mothers of children with NLE compared with sera from mothers of unaffected children (0.67 +/- 0.43 versus 0.14 +/- 0.30; P < 0.0001). There was a statistically significant difference in the mean anti-r-La levels between the sera of mothers of children with CHB compared with dermatologic NLE (0.51 +/- 0.45 versus 0.83 +/- 0.37 respectively; P = 0.0091). When we examined antibodies directed against the recombinant 52-kD Ro protein, there was a statistically significant elevation of titres in the sera of mothers of NLE children (0.77 +/- 0.35) compared with non-NLE mothers (0.29 +/- 0.39; P < 0.0001). There was no difference in the r-Ro levels between mothers of children with dermatologic NLE compared with CHB (0.82 +/- 0.37 versus 0.71 +/- 0.74; P = 0.32). When we examined polypeptides derived from the recombinant La protein, the mean number of polypeptides recognized by sera from mothers of children with NLE was significantly higher than the mean number of polypeptides recognized by sera from mothers of unaffected children (5.1 +/- 0.54 versus 2.3 +/- 0.54 respectively; P < 0.001). More importantly, when we examined the individual polypeptides, we found that only sera from mothers of children with NLE and not from mothers of unaffected children recognized a polypeptide designated DD (30% versus 0%, respectively). These studies indicate that the autoantibody response to the Ro/La particle can differentiate sera from mothers of children with NLE and sera from mothers of unaffected children. Furthermore, there was a difference in the anti-La autoantibody response between mothers of children with CHB and dermatologic NLE.(ABSTRACT TRUNCATED AT 400 WORDS)

The apoptosis-1/Fas protein in human systemic lupus erythematosus.

Three independent mutations involving the apoptosis-1 (APO-1)/Fas receptor or its putative ligand have led to lupuslike diseases associated with lymphadenopathy in different strains of mice. To determine whether humans with SLE also have a defect in this apotosis pathway, we analyzed the expression of APO-1 on freshly isolated blood mononuclear cells and on lymphocytes activated in vitro using flow cytometry and the monoclonal antibody anti-APO-1. Significantly higher level of APO-1 expression were detected on freshly isolated peripheral B cells and both CD4+ and CD8+ T lymphocyte populations obtained from lupus patients when compared with normal controls (P < 0.001). Almost 90% of the cells that stained positive for APO-1 also expressed the CD29 antigen, suggesting that APO-1 was upregulated after lymphocyte activation in vivo. No defect in APO-1 regulation was detected after activation of SLE T (with anti-CD3) or B (with Staphylococcus aureus Cowan 1) lymphocytes in the presence of IL-2 in vitro. Similarly, the anti-APO-1 antibody induced apoptosis in 74 +/- 5% of activated SLE T cells in vitro compared with 79 +/- 6% of the normal controls (P > 0.05). These results reveal that, while APO-1/Fas may play an important role in the regulation of lymphocyte survival in SLE, no consistent defect in the expression or function of the receptor could be detected in these studies.

Functional characterization of T cells bearing the gamma/delta T-cell receptor in patients with primary Sjögren's syndrome.

High percentages of gamma/delta+ T cells in the peripheral blood of a subgroup of patients with primary Sjögren's syndrome (SS) were found. This allowed us to purify and analyze them without their being previously expanded in vitro, and to investigate, therefore, the role of these cells in the pathological immune response which characterizes such systemic autoimmune disorders. The results showed poor proliferation of patient gamma/delta+ T cells in response to anti-CD3, due not to macrophage-dependent suppression but to defective interleukin 2 (IL-2) synthesis. Despite the defective proliferation patient gamma/delta+ cells, unlike those of the normal controls, provided a helper effect in inducing B cells to secrete immunoglobulins (Ig), particularly when they were preincubated with IL-2. The relative increase in a gamma/delta+ T cell subset which, although it secretes low levels of IL-2, is able to provide help for B-cell Ig synthesis, suggests that this T-cell subpopulation may be functional in vivo and may be involved in the pathological immune response encountered in pSS.

Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3.

Although proteinase 3 (PR3) has been identified as a major autoantigen in Wegener's granulomatosis, the precise antibody specificity(ies) and requirements for epitope recognition have not been characterized. We analyzed 11 sera containing antineutrophil cytoplasmic antibodies (cANCA) for binding to azurophilic granule proteins extracted from neutrophils under various conditions and for binding to native or rPR3. Ten of 11 (91%) of the cANCA sera bound to PR3 extracted by nonionic detergents when tested by immunoprecipitation or by IEF followed by capillary immunoblotting. Antibody binding to PR3 was retained when IEF was performed under dissociating conditions (8 M urea) indicating that PR3 is the major autoantigen in azurophilic granules and that association with other proteins is not required for antigenicity. In contrast, antigenicity was totally destroyed by exposure of PR3 to reducing agents or to low pH (less than 3.0) and was either lost or considerably diminished after boiling in SDS. cANCA sera also showed little or no binding to rPR3 expressed as a fusion protein in Escherichia coli or synthesized by wheat germ ribosomes in vitro. Inasmuch as PR3 enzymatic activity was partially retained after acid extraction, these findings indicate that cANCA bind to a limited number of conformational epitopes on PR3. In addition, IEF followed by capillary immunoblotting appears to be a sensitive and specific method to detect anti-PR3 antibodies in Wegener's granulomatosis.

[Effect of recombinant bovine somatotropin on the secretion and composition of milk in Sardinian ewes]. / Effetto della bST ricombinante sulla secrezione e sulla composizione del latte nella pecora sarda.

To verify the effect on the milk yield, 5 Sardinian ewes at 45 days from lambing were given two subcutaneous administrations, in a sustained-release vehicle, of recombinant bST in the dose of 3 mg/Kg of live weight, at 28 day intervals. Other 5 ewes of the same age, weight, distance from lambing and milk yield, were kept untreated. The observations lasted 56 days. The individual milk yield was recorded daily while both the milk (dry matter, ash, fat and protein) and some blood parameters (glucose, triglycerides, albumin, total protein, urea nitrogen, phospholipids, total and HDL cholesterol, calcium, phosphorus and magnesium) were checked weekly for each subject, by standard analytical methodology. The rbST significantly influenced the milk yield (+9.75% for the treated group) as already found in cows. The milk composition and the checked blood parameters were unaffected by the treatment.

[Hematologic parameters of the white donkeys of Asinara]. / Costanti ematiche dell'asinello bianco dell'Asinara.

In 14 albino donkeys (6 males and 8 females) aged 1-12 years, about 25% of the whole population, 30 blood parameters were determined, with automatic equipment, monthly, for 4 months. No significant differences between sexes or compared with other donkey breeds were found, in spite of the particular characteristics of the population and of their environment.

[Effect of the sudden removal of lambs on the blood levels of prolactin, triiodothyronine and thyroxine in ewes]. / Effetto dell'improvvisa sottrazione della prole sul livello ematico di prolattina, tri-iodotironina e tiroxina nella pecora.

The effect of the sudden removal of lambs, average age 42 days (36 to 54), on the blood level of PRL, T3 and T4 was checked, using a radioimmunological method, in 18 Sardinian ewes that had lambed in November (10 treated and 8 control). The two groups of animals, reared indoors in different sheep-folds, were subjected to the same management and feeding. Compared to day before the removal of the lambs, the blood PRL showed a sharp decrease which lasted 24 hours. After 4 days of observation, despite two daily milkings, the blood PRL had not returned to the initial levels. On the other hand, T3 and T4 showed an increase that lasted 48 hours, on the third day returning to the initial levels.

[The horses of Giara: variation of the thyroid activity and certain blood parameters between winter and summer]. / Il cavallino della Giara: variazioni dell'attività tiroidea e di alcune costanti ematiche tra l'inverno e l'estate.

The changes of some parameters in the blood serum of six Giara horses (3 males and 3 females) were checked weekly, in relation to the environmental temperature throughout a one winter and summer. T3, total lipids, triglycerides, urea nitrogen, creatinine, total protein, albumin, beta and gamma globulin showed significant difference between winter and summer and, with the exception of triglycerides, urea nitrogen and total protein were correlated to the environmental temperature. T4, glucose, uric acid and alpha globulin showed no difference between the two seasons and no correlation with the temperature. The results indicate that the seasonal variations in thyroid activity affected the T3 only, and that the blood lipids were transported from the beta globulin and, perhaps, in a smaller quantity, from the albumin, while the alpha globulin was not affected.