Circulating endothelial cell count: a reliable marker of endothelial damage in patients undergoing hematopoietic stem cell transplantation.

The physio-pathologic interrelationships between endothelium and GvHD have been better elucidated and have led to definition of the entity 'endothelial GvHD' as an essential early phase prior to the clinical presentation of acute GvHD. Using the CellSearch system, we analyzed circulating endothelial cells (CEC) in 90 allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients at the following time-points: T1 (pre-conditioning), T2 (pre-transplant), T3 (engraftment), T4 (onset of GvHD) and T5 (1 week after steroid treatment). Although CEC changes in allo-HSCT represent a dynamic phenomenon influenced by many variables (that is, conditioning, immunosuppressive treatments, engraftment syndrome and infections), we showed that CEC peaks were constantly seen at onset of acute GvHD and invariably returned to pre-transplant values after treatment response. Since we showed that CEC changes during allo-HSCT has rapid kinetics that may be easily missed if blood samples are drawn at pre-fixed time-points, we rather suggest an 'on demand' evaluation of CEC counts right at onset of GvHD clinical symptoms to possibly help differentiate GvHD from other non-endothelial complications. We confirm that CEC changes are a suitable biomarker to monitor endothelial damage in patients undergoing allo-transplantation and hold the potential to become a useful tool to support GvHD diagnosis (ClinicalTrials.gov NCT02064972).

Electroclinical evolution in ring chromosome 20 epilepsy syndrome: a case with severe phenotypic features followed for 25 years.

Intractable epilepsy and peculiar EEG patterns characterize ring chromosome 20 syndrome [r(20)], while dysmorphic features, mental retardation and behavioural disturbances are widely variable. The clinical evolution of r(20) over time is not well defined as relatively few cases have been reported. Here we describe a patient with severe clinical features followed for a 25-year period. The patient was subjected to clinical, psychometric and EEG evaluation twice a year from the age of 21 years. Cytogenetic studies, using chromosome analysis and fluorescence in situ hybridization (FISH) and several immunological investigations were performed. Ring chromosome 20 was found in 50% of examined metaphases with the deletion of subtelomeric regions 20p and 20q. Our patient presented with marked dysmorphic features, severe mental retardation, tetraparesis, dysarthria and intractable epilepsy with onset during the first year of life. During follow up, EEG findings and clinical features progressively worsened: a progressive disorganization of background EEG activity occurred and mental and motor impairment evolved. The severity of clinical expression depended on the extent of chromosomal deletion and on the haploinsufficiency of other important related genetic loci due to ring instability. The progressive worsening of both clinical and EEG features over a long period, which has also been reported by other authors, further characterized this syndrome.

Haploidentical peripheral blood and marrow stem cell transplantation in nine cases of primary immunodeficiency.

Bone marrow transplantation (BMT) is the treatment of choice in children affected by primary immunodeficiency (PID). Because only 10-15% of affected children have a familial HLA-identical donor alternative therapeutic options are BMT from a matched unrelated donor or an haploidentical BMT. In our experience only 40% of these children find a donor within the International Registry. Therefore, the remaining 50% children affected by PID are candidates for haploidentical BMT. Unfortunately, in PID other than sever-combined immunodeficiency (SCID), low engraftment rates have been reported because of minimal residual immunity. In order to enhance engraftment rate in haploidentical BMT in PID we suggest a protocol with addition of donor peripheral stem cells after mobilization with granulocyte colony-stimulating factor (G-CSF) (16 micrograms/kg for 5 days) and bone marrow cells. This procedure increases the cell load, which allows intensification of the conditioning regimen for induction of faster engraftment. The separation of CD34+ cells from leukapheresis products was achieved in the first 6 patients by the Isolex 300 system (Baxter) with a CD34+ cell purity range of 80-95% and in another three patients by the Clinimacs System (Miltenyi). The peripheral blood stem cells were cryopreserved until BMT, 15 days after G-CSF stimulation when the bone marrow was harvested, processed and T-cell depleted with Campath 1-M in the first 6 cases while the Clinimacs System was used in the remaining cases and no T-cell depletion was required. We included 9 patients in the study protocol: SCID (4), Omenn's syndrome (3), LAD (1) and CID (1). The mean value of peripheral CD34+ cells infused was 13.42 x 10(6)/kg and the mean CD3+ cells number was 0.385 x 10(5)/kg; the mean value of BM CD34+ cells infused was 10.62 x 10(6)/kg and the mean CD3+ cell number was 2.39 x 10(5)/kg. The mean number of infused CFU was 8.1 x 10(5)/kg for PBSC and 3.59 x 10(5)/kg for BM. The 9 patients achieved more than 0.5 x 10(9) peripheral blood neutrophils/L at a mean of 14.6 days (range: 6-22 days). One patient affected by SCID showed complete chimerism, but he died after BMT of systemic CMV infection; the other 8 patients are alive and well and 4 of them show complete chimerism in all cell lines. Split chimerism was documented in 2 SCID cases (CD3+ lymphocytes were of donor origin, monocytes were autologous and granulocytes were mainly autologous); 1 patient affected by Omenn's syndrome received 3 transplants (1 from the mother and 2 from the father, T-cells alone and bone marrow) and achieved engraftment with complete chimerism after the third transplant; the patient affected by LAD also received 3 transplants (2 bone marrow infusions and 1 PBSC infusion) achieving complete chimerism after the third one. In conclusion, the engraftment achieved in all treated patients, and the acceptable conditioning-related toxicity suggest that this approach could be successfully applied to children affected by PID and candidates for haploidentical BMT.

[Prenatal and postnatal transplantation of hematopoietic stem cells in children with primary immunodeficiency]. / Il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva.

Primary immunodeficiencies are inherited diseases characterized by impaired immune responses. In case of severe impairment of immunity bone marrow transplantation is the only therapeutic option. The molecular defect is known for several primary immunodeficiencies allowing prenatal diagnosis. This paper summarizes the clinical experience treating these pathologies by bone marrow transplantation.

Lower 24-h blood pressure regimen in subjects with a familial genealogy of longevity.

OBJECTIVE: This study investigates the 24-h blood pressure (BP) pattern in longevous subjects and their progeny, in order to validate the hypothesis that the human beings who live beyond their longest expectancy of life should be protected from developing hypertension. Such a characteristic feature is supposed to be a biological aspect of human longevity which can be transmitted to the progeny. MATERIALS AND METHODS: The study was carried out on 92 elderly subjects, (45 M, 47 W, 76-102 years), and 28 firstborn descendants of the first (7 M, 7 W, 36-55 years) and second (7 M, 7 W, 16-26 years) generation, in clinical health. The control subjects were 308 clinically healthy individuals (154 M, 154 F, 16-75 years) of the common population, stratified by age. RESULTS: The longevous subjects were found to show a diastolic daily mean level less pronounced than expected, according to the BP age-related trend in the common population. Both the children and grandchildren of the longevous subjects were seen to show a systolic and diastolic daily mean level significantly less pronounced than in their coeval subjects of the common population. CONCLUSIONS: Because of the unexpected lower diastolic BP daily mean level in the very old subjects, the hypothesis that the longevous subjects might be protected from developing hypertension via the arteriolar vasoconstriction seems to be confirmed. Because of the lower systolic and diastolic BP in young and adult subjects with a positive familiarity for longevity, the hypothesis that the hemodynamic protection from senile hypertension might be an inheritable biological feature of the longevity seems to be acceptable.

Continuous infusion of carboplatin during conventional radiotherapy treatment in advanced squamous carcinoma of the cervix uteri IIB-IIIB (UICC). A phase I/II and pharmacokinetic study.

OBJECTIVE: A prospective, single-arm phase-I/II trial performed to assess the efficacy and toxicity of the concomitant use continuous infusion of low-dose carboplatin and pelvic conventional radiotherapy in patients with locally advanced squamous cell carcinoma of the cervix. MATERIALS AND METHODS: Between January and July 1994, a total of 12 patients consecutively diagnosed to have squamous cell carcinoma of the cervix uteri stages IIB-IIIB UICC-TNM (five patients, IIB; and seven patients, IIIB) entered the study. All patients were evaluated by a gynecologist and a radiation oncologist and were submitted to standard pretreatment staging procedures. Radiation was delivered with 10-MeV photon beams with the shrinking-field technique. The patients received 2 Gy radiotherapy daily, 5 fractions per week, up to a planned total of 60 Gy in 6 weeks to the primary tumor and 46 Gy in 4 weeks to the whole pelvis. Irradiation was performed using four fixed orthogonal fields. One intracavitary insertion, 8 Gy to point A (dose rate, 1.1 Gy/h), was performed immediately after external pelvic irradiation. Carboplatin (12 mg/m2/day) was also administered in a continuous infusion, starting 1 day before the first fraction of radiotherapy. The platinum in plasma and urine, as well as the platinum concentration in the cytosols of lymphocytes and tumor, was measured weekly. RESULTS: A complete response was seen in nine (75%) of the 12 patients. Of the nine patients who achieved a complete remission, only one had subsequent failure in the pelvis. The total pelvic failure rate was 33.3% (four of 12 patients). With a median follow-up time of 20 months, the actuarial survival rate at 24 months was 64.8%. All patients completed the treatment without major protocol violations. Grade-2 leukopenia (in nine patients) and grade-1 nausea and vomiting (in five) were the most common acute toxicities. There was one grade-3 hematologic toxicity. Grade-3 late complications were observed in 16.6% of cases (two of 12 patients). On days 28 and 42 of the treatment, the mean total platinum plasma concentrations were 491 micrograms/L (SD = 129) and 672 micrograms/L (SD = 160), and the ultrafilterable fraction was 8-10%. At the same time points, the concentration in lymphocytes was constant at 21 picograms (pg) platinum/lymphocyte. The levels of platinum concentration measured on days 14 and 28 in the cytosols of tumor cells were 0.3 microgram/g (SD = 0.1) and 0.93 microgram/g (SD = 0.2). CONCLUSION: The combination of continuous infusion of carboplatin and radiotherapy at the aforementioned doses in patients with locally advanced cervical carcinoma resulted in a relatively low frequency of significant acute and late complications. Platinum in normal tissue (picograms per lymphocyte) was stable from week 1 of treatment, whereas the platinum steady state in plasma and in tumor cells was not reached in 6 weeks and was below that required in vitro to produce radiopotentiation. Further studies to determine the optimal dose of carboplatin and irradiation are needed prior to the initiation of phase-III studies.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

[The Campodimele study: 24-hour blood pressure in rural life style subjects]. / Studio di Campodimele: la pressione arteriosa di 24 ore in soggetti con stile di vita rurale.

The present paper is aimed at investigating the daily blood pressure in subjects with a rural style of life with the purpose of detecting whether or not the blood pressure regimen is influenced as expected because of the stress less prominent in the non-urban areas. Control data were obtained by the study of age- and sex-matching subjects with a metropolitan style of life. The results indicate the blood pressure has a lower daily level in rural subjects as compared to urban subjects. The daily baric impact is also lower, suggesting that the blood pressure regimen is really less pronounced in those who live according to a rural style of life. Such a lower magnitude allows us to experimentally suggest that the rural life is concrete in protecting the hemodynamic system from the higher level of blood pressure which are observable in subjects who live according to a metropolitan style of life. Interestingly, the expected phase anticipation in blood pressure circadian rhythm of rural subjects was not detected, as the wake up time was not so anticipated to act as a synchronizer.

Characterization of a distinct subpopulation of bovine gamma delta T cells.

A population of mononuclear cytotoxic cells from peripheral blood leucocytes of cattle showed no usual markers of B and T lymphocytes. However, it could be allocated to a previously unreported gamma delta T cell compartment. This assumption was suggested by: 1. The surface expression of CD3; 2. PCR amplification of the C delta TcR gene from cDNA; and 3. The detection of peripheral blood precursors expressing the workshop cluster (WC) 1 marker of bovine gamma delta T cells. These cells are recognized by murine monoclonal antibodies (mAbs) 5D4, 1E7, 6F9 and 8D7, raised in the authors' laboratory. The above mAbs also identify distinct groups of cells in thymus, spleen, lymph nodes and about 1% of uncultured PBL. The most diffuse infiltration of such cells was shown in the small intestine, as both intraepithelial and lamina propria lymphocytes. Mucosal homing activity was confirmed by immunoperoxidase staining on tongue and pharynx sections of healthy cattle.

Role of a distinct population of bovine gamma delta T cells in the immune response to viral agents.

A distinct population of bovine gamma delta T cells was isolated from peripheral blood mononuclear cells of foot-and-mouth disease (FMD)-vaccinated cattle; these lymphocytes were shown to exert a natural killer-like activity against cells infected by different viruses. The antiviral activity was dependent upon cognate recognition of target cells and could operate by both cytostatic and cytotoxic mechanisms. Among these, secretion of a serine esterase was shown after binding to target cells. This population of bovine gamma delta T cells is recognized by murine monoclonal antibodies 1E7, 5D4, and 6F9, raised in our laboratory. To define an in vivo antiviral role, four heifers were infected with a strain of bovid herpesvirus 1 by the intranasal/intravaginal routes and contact exposure. The prevalence of 1E7+/5D4+ cells among peripheral blood lymphocytes increased dramatically in the first days after infection; the same held true for in-contact cattle, albeit with a different time kinetics. In another experiment, colonization of mucosae was demonstrated by immunoperoxidase staining on tongue and palate sections of healthy cattle. The infiltration of gamma delta T cells altogether in the palate mucosa was much more accentuated in foot-and-mouth disease-vaccinated, as compared to nonvaccinated, control calves.

Campodimele study: blood pressure and heart rate pattern in clinically healthy elderly subjects.

Noninvasive ambulatory blood pressure (BP) monitoring is a developing method in clinical practice. Its interpretation needs reference standards stratified by age and gender. This study addresses ambulatory BP monitoring in elderly people with the purpose of quantifying the discrete and periodic variability of BP pattern over a 24-h period. The ABPM was performed in 92 clinically healthy subjects (45 men and 47 women) ranging in age from 76 to 102 years. The results refer to the time-qualified mean values with their dispersion, to the circadian rhythm with its parameters, and to the daily baric impact (BI) with its variability. The conclusion is drawn that BP preserves its nychtohemeral variability and circadian rhythmicity despite old age. The daily BP mean level and BI in older people in good health are comparable with those of young subjects, suggesting that humans surviving into old age are characterized by a eugenic control of their pressure regimen.

Production and characterization of monoclonal antibodies differentiating subpopulations of porcine B lymphocytes in blood and lymphoid tissues.

Monoclonal antibodies (mAbs) against various subclasses of immunoglobulin molecules are important reagents for the characterization and differentiation of serum immunoglobulins (Ig) generated during immune responses. Furthermore, Ig-specific mAbs can be powerful tools for the detection of B lymphocytes in blood and lymphoid organs. Here we describe two mAbs generated in our laboratories, named 1G6 and 2E8, which react with distinct epitopes on porcine immunoglobulin molecules. MAb 1G6 recognizes an epitope of an immunoglobulin chain with apparent molecular mass of 25 Kd. This chain represents an immunoglobulin light chain and might be the porcine equivalent of the murine and human kappa chain. MAb 2E8 is directed against porcine IgM molecules, recognizing an epitope of the porcine mu chain. The use of these mAbs was shown to avoid some common disadvantages of anti-Ig polyclonal antisera, like the high background staining of cells and tissue culture sections in immunohistochemistry. Furthermore, the use of these mAbs in two-color flow cytometry (FCM) versus a polyclonal anti-porcine Ig antiserum enables distinct B-lymphocyte subpopulations in blood and lymphoid organs to be detected. Our mAbs seem therefore to represent important and powerful reagents to identify and characterize porcine Ig isotypes and surface-Ig positive porcine B lymphocytes; their discriminating power between distinct B lymphocyte subpopulations could prove useful in different fields of both applied and fundamental immunological research.

New heteromyeloma cell lines for the production of human monoclonal antibodies.

The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.

Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells.

A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.

Serodiagnosis of Helicobacter pylori-associated gastritis with a monoclonal antibody competitive enzyme-linked immunosorbent assay.

Forty-nine monoclonal antibodies against Helicobacter pylori were screened to investigate their capacity to be used in enzyme-linked immunosorbent assay (ELISA) competitive systems for the serodiagnosis of Helicobacter pylori infection. On the basis of the inhibition pattern showed by the sera of five infected patients, the antibodies were subdivided into five groups. The immunoblotting analysis showed that the antibodies recognized a total of nine different antigenic determinants. In a study of the reaction of the antibodies with 12 isolates of H. pylori a total of 9 antigenic profiles were identified. Two monoclonal antibodies, HpN44 and HpN45, which recognized a 64-kD protein, were inhibited by all 5 positive sera. Antibody HpN45 was labeled with horseradish peroxidase, and the competitive ELISA was compared with an ordinary indirect ELISA in a study of 102 patients undergoing gastroscopy. Seventy-three patients proved to be infected by H. pylori according to urease or histologic tests. The sensitivity and specificity were 90.4% and 89.6%, respectively, for the indirect ELISA and 100% and 89.6% for the HpN45 competitive assay. The three patients who were 'false seropositive' with both serologic tests had atrophic gastritis. The high diagnostic performance and simplicity of the HpN45 monoclonal competitive ELISA make it suitable for routine serodiagnosis of H. pylori infection.

[A 24-hour blood pressure and heart rate pattern in healthy persons over 70 years of age. A Campodimele study]. / Comportamento di 24-h della pressione arteriosa e della frequenza cardiaca in vecchi sani ultrasettuagenari. Studio di Campodimele.

The present report is aimed at studying the 24-h pattern of blood pressure in elderly subjects (45 men and 47 women, aged from 76 to 102 years), recruited in Campodimele, a small city where the population of ultra septuagenaries is very dense. The investigation has been performed by mean of ambulatory non-invasive monitoring combined with chronobiometric estimates. The computation has been performed on raw data, rhythmic series and integral profiles. The analysis has been stratified according to sex. The tabulated estimates constitute an index of reference for blood pressure 24-h pattern in aged people.

[DNA index of colorectal carcinoma and follow-up]. / DNA index del carcinoma colon-rettale e follow-up.

In relation to DNA index we have studied 18 cases of differently evoluted carcinoma of the colon, some incoherently (8) other in agreement (10) with staging. Static and flow cytometric techniques have been employed, the first by means of densitometric study on paraffin sections with the Feulgen method, the second based on application of Hedley method on paraffin included material. DNA content, pressed by the DNA index, is inversely proportional to survival with similar results in both techniques, from which the possible prognostic significance is argued.

Antibodies for denatured human H-ferritin stain only reticuloendothelial cells within the bone marrow.

Human H-ferritin homopolymer was denatured in sodium dodecyl sulphate and injected in mice to obtain antibodies for dissociated H-subunit. The antisera and Moabs obtained were specific for the denatured H-chain with no cross-reactivity with assembled ferritins in immunoblotting experiments. In contrast the Moabs for native recombinant H-ferritin are specific for the assembled ferritin molecules with weak cross-reactivity with the denatured H-subunits. The epitope recognized by one of the anti-denatured H-chain Moabs was mapped on the C-terminal helix of ferritin. The antibodies were used to study H-ferritin conformation in cells. In immunocytochemistry experiments the antibodies for denatured H-ferritin stained HeLa and K562 cells weakly, with a different intensity and pattern to those obtained with anti-native H-ferritin antibody. In human bone marrow smears the anti-denatured ferritin antibodies stained only reticuloendothelial cells, and did not recognize the H-ferritin rich immature erythroblasts. It is concluded that assembled and denatured H-ferritins are immunogenically distinct, and that erythroid and reticuloendothelial cells within the bone marrow contain H-ferritin in different conformations.