Inhibition of Friend leukemia cell visceral metastases by a new monoclonal antibody and role of the immune system of the host in its action.

We developed a syngeneic mouse IgG2a monoclonal antibody (MAb) A9D41 directed against the Friend leukemia virus envelope gp70 antigen present on the cell surface membranes of virus producer 3C18 Friend leukemia cells (FLC). A9D41 showed a marked antitumor activity in DBA/2 mice given injections of gp70 positive 3C18 FLC, but it was ineffective in mice given injections of gp70 negative 745 FLC or unrelated tumor cells. A9D41 was particularly effective in inhibiting the development of 3C18 FLC liver and spleen metastases. MAb was also effective as adjuvant therapy in inhibiting visceral metastases after excision of an established s.c. FLC tumor, and combined therapy of A9D41 with mouse interferon alpha/beta was more effective than MAb or interferon alpha/beta alone. The immune system of the host played a decisive role in the antimetastatic action of A9D41. Thus, although MAb was cytotoxic for 3C18 FLC in vitro in the presence of rabbit complement, the F(ab')2 fragment was ineffective in vivo, and the antitumor effect of MAb was abolished in mice treated with an antibody to CD4 and diminished in natural killer cell-deficient beige and athymic nude mice. MAb-treated mice surviving injection of FLC developed an immune response to 3C18 FLC.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

A chromatin-associated protein is encoded in a genomic region highly conserved in the Plasmodium genus.

A single copy gene, pbB7, encoding a putative 26 kDa acidic protein has been isolated from Plasmodium berghei and appears to be part of a genomic region well conserved within the Plasmodium genus. The deduced amino acid sequence exhibits significant blocks of similarity with nucleosome assembly proteins from yeast and man. The nuclear localization of the natural protein and its close association with chromatin during the entire erythrocytic cycle of the parasite have been demonstrated using specific monoclonal antibodies against the pbB7 product expressed in Escherichia coli. These results suggest an involvement of this nuclear factor in the dynamics of chromatin packaging.

Intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT): a nuclear magnetic resonance study on T-lymphoblastoid cell lines with different resistance to AZT.

This paper reports the results of 31P and 1H nuclear magnetic resonance (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deoxythymidine (AZT) in the human CD4+ T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated by prolonged culturing of CEM cells in the presence of increasing AZT concentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected in the sensitive cell line in concentrations above the NMR detection levels. In another cell line, MOLT-4, which is less sensitive to AZT effects, the intracellular level of AZT-MP was much lower and was only slightly raised by increasing the concentration of AZT in the extracellular and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activity with respect to the parental clone, the intracellular AZT-MP concentration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 extracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in these cells. The possible effects of additional mechanisms of drug resistance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiretroviral nucleoside analogues.

Pentose phosphate pathway alterations in multi-drug resistant leukemic T-cells: 31P NMR and enzymatic studies.

31P NMR studies were carried out on the parental drug-sensitive human T-lymphoblastoid cell line CCRI-CEM (CEM) and its multi-drug-resistant (MDR) CEM-VBL100 variants, to assess the role of the pentose phosphate (PP) in MDR expression. CEM and CEM-VBL100 were incubated in the presence of 2-deoxyglucose, as recently proposed by our group (Clin. Chim. Acta 208: 39, 1992). Accumulation of 2-deoxyglucose 6-phosphate was much lower in the drug-resistant than in sensitive cells, indicating PP shunt activation in the MDR variants. This result was confirmed by enzymatic analyses, which demonstrated that, with respect to the parental line, the MDR variant was characterized by a) unaltered hexokinase activity; b) higher glucose 6-phosphate dehydrogenase activity; c) increased levels of reduced glutathione and marked increase of glutathione peroxidase activity after cell exposure to an oxidizing agent (tert-butylhydroperoxide). These results support the view that cell detoxification mechanisms mediated by the pentose phosphate pathway may contribute to the expression of MDR in tumours.

High frequency production of hybridomas secreting antibodies to cell antigens.

The efficiency of various immunization protocols for the production of hybridomas secreting immunoglobulins specific to cell antigens was evaluated in 15 different fusion experiments. Some of these experiments were performed with splenic B-lymphocytes from mice at different stages of immunization. This approach allowed a dynamic analysis of immunocompetent splenic B-lymphocyte production during the immunization cycle.

[Schönlein-Henoch syndrome: clinical-epidemiological analysis of 98 cases]. / Sindrome di Schönlein-Henoch: analisi clinico-epidemiologica su 98 casi.

We analyzed the clinical and epidemiological data of 98 patients (50 males and 48 females) aged between 6 months and 16 years, hospitalized for Schönlein-Henoch syndrome in the last 20 years. The incidence was higher during spring time. Throat culture was positive for streptococcus pyogenes in 16% of patients. 14 of 60 (23%) had a positive allergometric response to various tests. The extrarenal manifestations were: purpura (100%), articular (68%) or gastrointestinal (32%) involvement and orchitis (10%). Renal symptoms were observed in 23% of the patients, but a clear nephropathy was documented just in 5% of the cases, with resolution within 2 years. Our data suggest, in agreement with the letterature, that renal involvement in Schönlein-Henoch syndrome has usually a benign course.

Inflammatory bowel disease in children and adolescents in Italy: data from the pediatric national IBD register (1996-2003).

BACKGROUND: The purpose was to assess in Italy the clinical features at diagnosis of inflammatory bowel disease (IBD) in children. METHODS: In 1996 an IBD register of disease onset was established on a national scale. RESULTS: Up to the end of 2003, 1576 cases of pediatric IBD were recorded: 810 (52%) ulcerative colitis (UC), 635 (40%) Crohn's disease (CD), and 131 (8%) indeterminate colitis (IC). In the period 1996-2003 an increase of IBD incidence from 0.89 to 1.39/10(5) inhabitants aged <18 years was observed. IBD was more frequent among children aged between 6 and 12 years (57%) but 20% of patients had onset of the disease under 6 years of age; 28 patients were <1 year of age. Overall, 11% had 1 or more family members with IBD. The mean interval between onset of symptoms and diagnosis was higher in CD (10.1 months) and IC (9 months) versus UC (5.8 months). Extended colitis was the most frequent form in UC and ileocolic involvement the most frequent in CD. Upper intestinal tract involvement was present in 11% of CD patients. IC locations were similar to those of UC. Bloody diarrhea and abdominal pain were the most frequent symptoms in UC and IC, and abdominal pain and diarrhea in CD. Extraintestinal symptoms were more frequent in CD than in UC. CONCLUSIONS: The IBD incidence in children and adolescents in Italy shows an increasing trend for all 3 pathologies. UC diagnoses exceeded CD.

Expression of lymphocyte homing receptor gene is lost in multi-drug-resistant variants of human T-lymphoblastoid CCRF-CEM cells.

The 2.2-kb human cDNA clone PBL32, encoding for the lymphocyte homing receptor (LHR) was used to study the expression of this determinant in multi-drug-resistant (MDR) variants of human T-lymphoblastoid CCRF-CEM (CEM) cells. LHR is significantly associated with the drug-sensitive phenotype, its expression being progressively and quantitatively reduced in MDR variants of CEM cells according to the extent of drug resistance.

Association of the dystroglycan complex isolated from bovine brain synaptosomes with proteins involved in signal transduction.

Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.

Cloning and expression of a cDNA encoding an elongation factor 1beta/delta protein from Echinococcus granulosus with immunogenic activity.

A cDNA clone (Eg EF-1beta/delta) of Echinococcus granulosus has been isolated by an expression library screened with immunoglobulin (Ig)E of sera from patients with cystic echinococcosis (CE). The Eg EF-1beta/delta was identified on the basis of sequence homology to the subunits beta or delta of the elongation factor-1. The amino acid sequence deduced from this open reading frame is 244 residues long with a predicted molecular mass of 31 kDa. In Southern blot under high stringent condition, Eg EF-1beta/delta hybridized to genomic DNA of E.granulosus at two bands of 4 and 2.5 Kb. In immunoblotting analysis, the Eg EF-1beta/delta protein shows immunological reactivity with sera from CE patients: 51.7% of sera contained IgE, 41.7% IgG and 18.3% IgG4 specific to the recombinant protein. We identify the Eg EF-1beta/delta by immunoblotting with specific monoclonal antibody both in protoscoleces and in sheep hydatid fluid. The higher percentage of humoral immune response against Eg EF-1beta/delta observed in CE patients with calcified cysts than in patients with active cysts indicates the possible release of the protein in the hydatid fluid after protoscoleces degeneration suggesting the possible use of this antigen in the immunosurveillance of CE. Overall, these findings seem to assign to Eg EF-1beta/delta a key role in the allergic disorders and in the complex host-parasite relationship in CE.

Long-term cultures of human fetal liver cells: a three-dimensional experimental model for monitoring liver tissue development.

BACKGROUND/AIMS: The present study describes an embryonic-fetal liver culture system which allows morphogenetic interactions consistent with the development of the hepatocellular function. METHODS: Intact livers from 8-12-week embryos were soaked in an extracellular matrix at 4 degrees C and gently dissociated without any enzymatic treatment. The resulting spherical hepatic units were cultured in a chemically defined serum-free medium and seeded into an extracellular matrix layer. Adherent three-dimensional tissue specimens were examined at various times by light and electron microscopy to evaluate the maintenance of hepatocyte morphology. RESULTS: The liver cells were viable for over 4 months; erythropoietic burst colonies were detected for longer than 6 weeks. Parallel detection of bile salt production in the medium by high performance liquid chromatography proved liver tissue functionality. Bile salt composition revealed predominance of taurine-conjugates rather than glycine. Maximum bile salt concentration (approximately 3 months) coincided with structural and ultrastructural observations indicating a marked decline in hematopoiesis, well-defined biliary canaliculi and formation of an organ-like structure. CONCLUSIONS: This three-dimensional culture system recapitulates fetal liver development with: (i) initial proliferation of both fetal erythropoietic and hepatic cells and (ii) subsequent shut-off of erythropoiesis and a shift to a more advanced stage of hepatocyte function, such as bile salt secretion.

Native and recombinant antigens in the immunodiagnosis of human cystic echinococcosis.

To evaluate the diagnostic sensitivity and specificity of immunoelectrophoresis (IEP), indirect haemagglutination (IHA), enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB), we compared their ability in detecting IgG antibodies to a hydatid fluid fraction (HFF) and to native and recombinant antigen B of Echinococcus granulosus. We tested sera from patients who had cystic echinococcosis (CE) grouped according to their type of cysts (n = 204), from patients with other parasitic diseases (n = 21), lung or liver carcinomas (n = 6) or serous cysts (n = 26) and from healthy controls (n = 90). HFF-IB gave the highest sensitivity (80%) followed by ELISA (72%), IHA (54%) and IEP (31%), respectively. The diagnostic sensitivity significantly (P < 0.01) decreased as cysts matured from type I-II to type VII. Recombinant and native antigen B-IB yielded similar sensitivity (74%). A large number of clinically or surgically confirmed CE patients (20%) resulted negative. In these patients' sera, IB to assess the usefulness of the recombinant E. granulosus elongation factor-1 beta/delta in detecting IgE antibodies yielded 33% of positivity. Our findings underline the need to standardize techniques and antigenic preparations and to improve the performance of immunodiagnosis by characterizing new antigens and detecting distinct immunoglobulin classes.

Pneumocystis carinii infection in young non-immunosuppressed rabbits. Kinetics of infection and of the primary specific immune response.

The aim of this study was to determine the kinetics, the dissemination of the infection and the immunological response to Pneumocystis carinii primary infection in a non-immunosuppressed rabbit model. For this purpose, we developed a nested PCR that amplified a portion of the mitochondrial large-subunit rRNA gene of rabbit-derived P. carinii. The PCR detected P. carinii DNA in lung and bronchoalveolar lavage fluids from 14- to 45-day-old rabbits but not in their serum. No P. carinii DNA was detected in extrapulmonary organs from 28-day-old rabbits with P. carinii pneumonia. ELISA and immunoblotting analysis showed that 5-day-old pups had elevated specific IgG. The IgG concentration sharply decreased, reaching a trough on day 21, and from then onwards progressively increased as the infection cleared. Conversely, the specific IgM concentration increased during the infection and peaked on day 28. IgG mainly recognized a 50-kDa subunit of P. carinii organisms; IgM recognized first a 45-kDa subunit on day 21, whereas from day 28 onwards it also recognized the 50-kDa subunit. A P. carinii-specific splenocyte proliferative response was observed on day 45. These findings suggest that P. carinii primary infection is a time-limited and a lung-limited event and contribute new information on the relationship between the kinetics of primary P. carinii infection and the immunological response in a model that mimics the primary infections in humans.

Murine monoclonal antibody recognizing a 90-kDa cell-surface determinant selectively lost by multi-drug-resistant variants of CEM cells.

We describe a murine IgG1 monoclonal antibody (MAb56), specific for a cell-surface protein structure (MC56 determinant) expressed by the human CEM cell line. A large band of approximately 90 kDa was identified as the main specific component of the MC56 determinant. Such a 90-kDa protein is significantly associated with the drug-sensitive phenotype, its expression being progressively reduced quantitatively in multi-drug-resistant (MDR) variants of CEM cells, according to the extent of drug resistance. In addition, the MC56 determinant is expressed de novo in drug-sensitive revertant cell lines derived from MDR cells and unreactive with the MAb56. The MAb56 shows a high affinity towards the immunizing drug-sensitive CEM cell line (Ka = 1.86 x 10(9) L/mole) while not binding to MDR cell variants. The expression of the MC56 molecule on a variety of human cells and tissues makes such a cellular determinant a candidate as a marker for studying the MDR phenomenon both in vivo and in vitro.

Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants.

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.