Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Successful xenografting of cryopreserved primary pancreatic cancers

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.     

Scanning electron microscopic characterization of healing and normal rat ligament microstructure under slack and loaded conditions

The objective of this study was to observe and compare behavior of the collagen fiber microstructure in normal and healing ligaments, both in situ and ex vivo, in order to add insight into the structure-function relationship in normal and healing ligaments. Fifty-two ligaments from 26 male rats were investigated. Eleven animals underwent surgical transection of both medial collateral ligaments (MCLs) (22 ligaments), which were allowed to heal for a period of 2 weeks. An additional 15 animals (30 ligaments) were used as normals. Ligaments were placed into six groups: Slack (n = 6 control, n = 6 healing), Reference (n = 4 control, n = 4 healing), Loaded (n = 4 control, n = 4 healing), 15 degrees Flexion (n = 4 control, n = 4 healing), 120 degrees Flexion (n = 4 control, n = 4 healing), and Tissue Strain vs. Flexion Angle (n = 8 normals). All ligaments, except those in the Tissue Strain vs. Flexion Angle group, were prepared for scanning electron microscopy. Tissues were harvested, mounted in a load frame, and chemically fixed in one of five states: (1). slack, (2). reference (onset of loading), (3). loaded, (4). 15 degrees knee flexion, or (5). 120 degrees knee flexion. After fixation the tissues were prepared for electron microscopy (SEM). The micrographs from the slack, reference, and loaded groups show fiber straightening with loading in normal ligaments as well as in both scar and "retracted" regions of healing ligaments. Collagen fibers' diameter and crimp patterns were dramatically changed in the scar region of healing ligaments: Width decreased from 19.4 +/- 1.7 microm to 6.5 +/- 2.1 microm (p <.000001), period from 51.4 +/- 15.1 microm to 11.0 +/- 2.4 microm (p <.000001), and amplitude from 9.8 +/- 0.8 microm to 3.9 +/- 0.8 microm (p <.000001). Normal ligaments fixed in situ show wavy regions at 120 degrees but less so at 15 degrees flexion. Healing ligaments fixed in situ show regions of fiber waviness in the scar region at 120 degrees and also at 15 degrees flexion, indicating ligament laxity persists toward both extremes of the range of motion. The data suggest that straightening of crimped fibers is a functionally relevant phenomenon, not only in normal but also in healing ligaments.     

Dexamethasone and clenbuterol detection by enzyme immunoassay in Bovine Liver Tissue: A new multiresidue extraction procedure

A fast and simple extraction procedure was developed for simultaneous determination in bovine liver of two veterinary drugs, widely used as growth promoters in meat production: dexamethasone (a synthetic corticosteroid drug) and clenbuterol (a beta2‐adrenergic agonist drug). Liver samples were extracted by acetonitrile, without any clean‐up step. Two different ELISAs, specific for the two classes of drugs, were used to determine the residue concentration in the extracts. The intra‐ and inter‐extraction variability was determined at different concentrations: the intra‐extraction coefficients of variation (CVs) were between 2.5 and 17.7% for dexamethasone and between 0.9 and 9.8% for clenbuterol; the inter‐extraction CVs were between 2.0 and 16.8% for dexamethasone and between 0.5 and 10.8% for clenbuterol. Recovery ranged from 92 to 154% for dexamethasone and from 78 to 105% for clenbuterol. The limit of detection was 1.43 ng g^?1 and 0.43 ng g^?1, respectively. The limit of quantification for dexamethasone was 2.09 ng g^?1 and for clenbuterol was 0.72 ng g^?1. The combination of the new extraction procedure with an ELISA detection permitted the rapid semi‐quantitative determination of both dexamethasone at its maximum residue level (MRL: 2.5 ng g^?1 in liver tissue), and clenbuterol at low concentration level.     

mdm2 gene alterations and mdm2 protein expression in breast carcinomas

This study investigates the mdm2 gene status and expression in 66 surgically resected human breast carcinomas, with correlations with clinico-pathological and biological data (histological type, grading, steroid receptor status, p53 expression, proliferative activity). Four (7.7 per cent) out of 52 informative cases bear mdm2 gene amplification (four- to ten-fold) and 8 (15.4 per cent) of 52 cases showed borderline amplification (three-fold). Nine (13.6 per cent) out of 66 cases showed strong mdm2 nuclear immunoreactivity. Twenty-seven (40.9 per cent) cases showed isolated mdm2 reactive nuclei. All cases with clear amplification showed a high percentage of mdm2 immunoreactive nuclei. The relationship between gene amplification and mdm2 protein expression is highly significant (P<0.0001). No association was observed between mdm2 gene amplification and any of the considered clinico-pathological and biological parameters, while mdm2 immunoreactivity showed a significant association only with oestrogen receptor immunoreactivity (P=0.009). p53 expression was associated neither with mdm2 gene amplification nor with mdm2 immunoreactivity. It could be tempting to hypothesize that the evaluation of the combined mdm2/p53 immunohistochemical phenotype in human breast carcinoma could give us better prognostic information than the evaluation of the expression of the p53 protein alone.     

Etherification of poly(oxymethylene)-diol [α-hydro-ω-hydroxypoly(oxymethylene)] with orthoesters in the presence of Lewis acids

Attempts were made to elucidate experimentally the behaviour of poly(oxymethylene)-diol [α-hydro-ω-hydroxypoly(oxymethylene)] in the etherification reaction in order to explain the decrease of the polymer molecular weight compared with that of the starting product and in order to determine the type of terminal groups and how the thermal resistance and alkali stability can be affected. In fact one finds that, in addition to the wanted terminal ether groups, formyl and orthoformyl (dialkoxymethyl) groups are formed having a different chemical resistance in comparison to that of the pure ethers, particularly in basic media.     

Cystometric changes in the early phase of streptozotocin-induced diabetes in rats: evidence for sensory changes not correlated to diabetic neuropathy

Summary 1. The effect of streptozotocin (STZ) induced diabetes on rat urinary bladder function was investigated by means of in vivo cystometry and in vitro recording of bladder strips contractility. A group of sucrose-fed animals was included to determine to what extent the STZ-induced changes were ascribable to the increased diuresis. 2. After 7–9 weeks from STZ injection there was a marked increase in weight of bladder and ureters. Cystometry revealed a marked increase in bladder capacity (volume threshold) although pressure threshold and amplitude of micturition contraction were unaffected. Sucrose-fed animals, having normal blood glucose levels but a similar increase in urine production exhibited cystometric changes identical to those of STZ animals. 3. In vitro experiments indicated that the response to field stimulation (0.1–20 Hz) is reduced in STZ-pretreated but increased in sucrose-fed animals, as compared to controls. 4. The content of urinary bladder and ureters in sensory neuropeptides (substance-P, neurokinin-A and calcitonin-gene related peptide-like immunoreactivity) was increased by STZ diabetes when values were corrected for the increased weight of these organs. 5. The capsaicin-induced contraction of the rat isolated bladder strips, presumably caused by neuropeptides released from intramural sensory nerves, is unaffected by STZ diabetes. 6. These findings indicate that STZ diabetes produces, at an early stage, changes similar to those reported to occur in the human disease, e. g. a greater bladder capacity with unimpaired voiding function. The increased bladder capacity of STZ-rats seems largely, if not solely, ascribable to changes in physical properties of the detrusor muscle, thereby allowing accomodation of greater than normal volumes with similar increase of intraluminal pressure. No sign of diabetic neuropathy of the capsaicin-sensitive sensory nerves can be observed at this stage (7–9 weeks) of STZ diabetes. Send offprint requests to P. Santicioli at the above address