Combined therapeutic effect of a monoclonal anti-idiotype tumor vaccine against NeuGc-containing gangliosides with chemotherapy in a breast carcinoma model.

Anti-idiotypic monoclonal antibodies (mAb) have been evaluated for actively induced immunotherapy with encouraging results. However, rational combination of cancer vaccines with chemotherapy may improve the therapeutic efficacy of these two approaches used separately. The main objective of this study was to evaluate the antitumor effect of the co-administration of 1E10 (Racotumomab), a monoclonal anti-idiotype tumor vaccine against an IgM mAb, named P3 that reacts specifically with NeuGc-containing gangliosides and low-dose Cyclophosphamide in a mammary carcinoma model. F3II tumor-bearing mice were immunized subcutaneously with 100 microg of 1E10 mAb in Alum or with 150 mg/m(2) of Cyclophosphamide intravenously 7 days after the tumor inoculation. While a limited antitumor effect was induced by a single 1E10 mAb immunization; its co-administration with low-dose Cyclophosphamide reduced significantly the F3II mammary carcinoma growth. That response was comparable with the co-administration of the standard high-dose chemotherapy for breast cancer based on 60 mg/m(2) of Doxorubicin and 600 mg/m(2) of Cyclophosphamide, without toxicity signs. Combinatorial chemo-immunotherapy promoted the CD8(+) lymphocytes tumor infiltration and enhanced tumor apoptosis. Furthermore, 1E10 mAb immunization potentiated the antiangiogenic effect of low-dose Cyclophosphamide. Additionally, splenic myeloid cells Gr1(+)/CD11b(+) associated with a suppressor phenotype were significantly reduced in F3II tumor-bearing mice immunized with 1E10 mAb alone or in combination with low-dose Cyclophosphamide. This data may provide a rational for chemo-immunotherapy combinations with potential medical implications in breast cancer.

Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and antiangiogenic effect in a metastatic lung carcinoma.

Anti-idiotype monoclonal antibody (mAb) 1E10 was generated by immunizing BALB/c mice with an Ab1 mAb which recognizes NeuGc-containing gangliosides, sulfatides and some tumor antigens. 1E10 mAb induces therapeutic effects in a primary breast carcinoma and a melanoma model. However, the tumor immunity mechanisms have not been elucidated. Here we show that aluminum hydroxide-precipitated 1E10 mAb immunization induced anti-metastatic effect in the 3LL-D122 Lewis Lung carcinoma, a poorly immunogenic and highly metastatic model in C57BL/6 mice. The therapeutic effect was associated to the increment of T cells infiltrating metastases, the reduction of new blood vessels formation and the increase of apoptotic tumor cells in lung nodules. Interestingly, active immunization does not induce measurable antibodies to the 1E10 mAb, the NeuGc-GM3 or tumor cells, which may suggest a different mechanism which has to be elucidated. These findings may support the relevance of this target for cancer biotherapy.

[De novo heterozygous mutation in the MBD5 gene associated with bilateral band heterotopia and polymicrogyria]. / Mutación de novo en heterocigosis en el gen MBD5 asociada a heterotopía en banda bilateral y polimicrogiria.

INTRODUCTION: The aetiology of autosomal dominant mental retardation type 1, also known as pseudo-Angelman, MBD5-associated neurodevelopmental disorder or MBD5 haploinsufficiency, lies in a microdeletion of chromosome 2q23.1 or in a specific alteration of the MBD5 gene, which constitutes the minimum region affected in the aforementioned microdeletion. AIM: To report the case of a girl with a heterozygous de novo mutation in the MBD5 gene associated with bilateral band heterotopia and polymicrogyria. CASE REPORT: We report the case of an 8-year-old girl who was submitted to a developmental follow-up from the age of 18 months after presenting the association of severe intellectual disability and motor delay, lack of language development, segmental hypotonia, a wide forehead and kyphoscoliosis. Magnetic resonance imaging of the brain revealed the presence of a bilateral band heterotopia and parietooccipital polymicrogiria predominant on the left side. In the exome the de novo heterozygous variant c.397+1G>C was detected in the MBD5 gene. CONCLUSION: This is the first observation of a heterozygous mutation in the MBD5 gene associated with a neuronal migration disorder.

TITLE: Mutación de novo en heterocigosis en el gen MBD5 asociada a heterotopía en banda bilateral y polimicrogiria.Introducción. La etiología del retraso mental autosómico dominante 1, también conocido como pseudo-Angelman, trastorno del neurodesarrollo asociado a MBD5 o haploinsuficiencia MBD5, radica en una microdeleción del cromosoma 2q23.1 o en una alteración específica del gen MBD5, que constituye la mínima región afectada en la citada microdeleción. Objetivo. Comunicar el caso de una niña con una mutación heterocigota y de novo en el gen MBD5 asociada a heterotopía en banda bilateral y polimicrogiria. Caso clínico. Niña de 8 años, seguida evolutivamente desde los 18 meses por presentar la asociación de discapacidad intelectual y retraso motor graves, ausencia de desarrollo del lenguaje, hipotonía segmentaria, frente ancha y cifoescoliosis. En la resonancia magnética cerebral se observó la presencia de una heterotopía en banda bilateral y polimicrogiria parietooccipital de predominio izquierdo. En el exoma se detectó la variante de novo c.397+1G>C en heterocigosis en el gen MBD5. Conclusión. Constituye la primera observación con una mutación heterocigota en el gen MBD5 asociada a un trastorno en la migración neuronal.

Antitumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides.

We examined the antitumor effects of 1E10 monoclonal antibody, an anti-idiotypic IgG to an IgM monoclonal antibody, named P3, that reacts specifically with N-glycolyl-containing gangliosides and also recognizes antigens in human breast and melanoma tumors. Two murine tumor cell lines positive for the P3 antibody, F3II mammary carcinoma (BALB/c) and B16 melanoma (C57BL/6), were employed. In BALB/c mice, vaccination with several i.p. doses at 14-day intervals of 50 microgram of 1E10 coupled to keyhole limpet hemocyanin in Freund's adjuvant, significantly reduced s.c. tumor growth of F3II carcinoma cells and the number of spontaneous lung metastases. Also, the effect of 1E10 as a biological response modifier on tumor lung colonization was evaluated in C57BL/6 mice injected i.v. with B16 melanoma cells. Interestingly, i.v. administration of 10 microgram of uncoupled 1E10 antibody, 10-14 days after inoculation of B16 cells, dramatically reduced the number of experimental metastases in comparison with lungs from mice treated with an irrelevant IgG. The present data suggest that this 'non-internal image' anti-idiotypic monoclonal antibody may activate more than one mechanism of antitumor response against melanoma and mammary tumor cells.

Somaclonal variation in rye.

We studied the genetic variation generated during in vitro culture of rye Secale cereale L. We analyzed the progenies of four generations of the plants regenerated from immature embryo cultures. A high frequency of mutant plants was observed, 50.75%, this frequency was genotype dependent. Other characteristics typical of somaclonal variation were also observed: the obtaining of dominant mutations, the presence of more than one mutation per plant, the obtaining of homozygous mutants and a high rate of mutation of particular loci. In some cases transposable elements could be implicated. We postulate that tissue culture could induce mutations as well as select particular cell types and so increase the appearance of special mutants.

Immunogenetic analysis of variable regions encoding AB1 and gamma-type AB2 antibodies from the NeuGc-containing ganglioside family.

The variable regions from P3, a murine monoclonal antibody (MAb) against NeuGc-containing gangliosides, and two anti-idiotype MAbs directed to P3 MAb were cloned and sequenced. Comparisons with previously reported sequences showed that P3 is a germline antibody encoded by genes from the V(H)Q52 and V(kappa)19 families. Analysis of nucleotides at the heavy chain CDR3 (H-CDR3) showed the presence of an extensive 3' N region that contains almost 50% of the nucleotides of this CDR. In addition, amino acid sequence analysis of the H-CDRs of this MAb revealed the presence of three arginines, two of which are present in the H-CDR3, that could be involved in the interaction of P3 MAb with its electronegative epitope on gangliosides. Anti-idiotype 1E10, which seems to define a "regulatory" idiotope on P3 MAb (it induces Id+ Ab3), represents a germline Ab2 that belongs to the V(H)J558 and V(kappa)10 gene families. By contrary, the anti-idiotype 3B11 is an extensively mutated antibody that belongs to the V(H)3660 and V(kappa)4/5 gene families, defining a "private" idiotope on P3 MAb. Even when different V genes contribute to the variable regions of 1E10 and 3B11 MAbs, they share an acidic motif E/D-D-Y/D-Y-D in H-CDR3, suggesting that both Ab2s recognize paratope positive residues on the Ab1. Therefore, complementary electrostatic interactions involving H-CDR3 from both Ab1 and Ab2, might provide a clue to understand the molecular basis for the generation of gamma-type anti-idiotype antibodies to V regions recognizing glycolylated ganglioside antigens.

Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody.

An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

Characterization of the colorectal antigen IOR-C2.

A colorectal antigen (IOR-C2) was characterized by a monoclonal antibody produced against the colon cancer cell line SW1116. By immunohistochemical staining the antigen was abundant and strongly expressed in epithelium of normal colon whereas colorectal carcinomas showed a more variable and heterogenous reactivity to the antibody (IOR-C2). Radioimmunoprecipitates of SW1116 cell homogenates showed a 160-200 kD band in SDS gels. Physicochemical characterization indicate that at least two IOR-C2 reactive sites are present on the antigen tested and that it is mainly an 0-linked glycoprotein carbohydrate chain which can also be N-linked to the protein. The expression of IOR-C2 mimics that of the colon associated antigen (CAA) and NCC-CO-450 antigen but is distinct from these with regard to its expression in carcinomas as well as its physicochemical characteristics.

T cell-independent B cell response to self-monosialogangliosides: primary response monoclonal antibodies.

Purified GM1 and GM2 gangliosides incorporated into liposomes were injected subcutaneously in BALB/c mice every 3-4 days after pretreatment of the animals with low-dose cyclophosphamide. Serum samples were collected at different intervals and tests by ELISA for the presence of anti-ganglioside antibodies. Four doses (50 micrograms each) were sufficient to raise a measurable primary type of response to GM1, while nine doses were required to obtain measurable IgM antibody titers to GM2. Three monoclonal antibodies (MAbs) wer generated by fusing splenocytes with mouse myeloma cells. The specificity of MAbs was determined by ELISA and HPTLC-immunostaining using a panel of purified glycolipids. The MAb designated E1 showed a high degree of specificity because it reacted only with N-acetyl GM2. Monoclonal antibody A3 reacted predominantly with GM2 and GM1, but also reacted moderately with the GM3 ganglioside. The epitope recognized by this MAb is suggested to be the trisaccharide sequence GalNAc beta 1-4(NeuAc alpha 2-3)Gal. The third MAb (F6) reacted strongly with GM1 but a weak reactivity was also observed with GD1b as well as with asialo-GM1, indicating that the terminal tetrasaccharide Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal- structure is probably involved in antigenic recognition. Formalin-fixed and paraffin-embedded tissue sections were stained with the E1 and A3 MAbs, using the avidin-biotin complex (ABC) technique. Strong immunoreactivity for E1 appeared in the tumor cells of five primary lung carcinomas and in five malignant melanomas. No immunoreactivity was demonstrated in the parenchyma of a lung without malignancy, or in a metastasis from a colon carcinoma.

Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that also recognizes sulfated glycolipids.

The P3 murine monoclonal antibody (MAb) was generated by immunizing BALB/c mice with NeuGcGM3 included into liposomes. The specificity of this MAb was defined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms. P3 MAb binds to NeuGc-containing gangliosides and was shown also to react with sulfated glycolipids. A preliminary immunohistochemical study showed that the P3 MAb was able to recognize antigens expressed in human breast tumors.

A mouse IgG1 monoclonal antibody specific for N-glycolyl GM3 ganglioside recognized breast and melanoma tumors.

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund's adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

A medical information system for ambulatory care, research, and curriculum in an army family practice residency: 51,113 patient problems.

Over a 16-month period, the Family Practice Residency at Madigan Army Medical Center coded 51,113 ambulatory problems using the International Classification of Health Problems in Primary Care (ICHPPC) coding scheme. The demography of this defined active duty, retired, and dependent population is described as well as the rank order of the major diagnostic categories and top 25 individual diagnoses. A plot of the major diagnostic categories seen by month over a 16-month period is presented. Comparisons between similar curves are made. Physician compliance to coding approached 75 percent and an average of 1.42 problems were dealt with (coded) per visit. Fifty percent of all patient problems recorded fell into 25 descriptive diagnoses. Ninety percent of all patient problems recorded fell into 163 descriptive diagnoses. These data compare favorably to the Virginia study. Comparison of the top 25 patient problems also revealed similarities between this and the Virginia study. Difficulties arise in comparing data because of the differences in coding schemes (RCGP vs ICHPPC). A universally adopted code for all family practice and ambulatory care research would help in overcoming this problem.

[Pilot study with the monoclonal antibody IOR-C5 as a potential agent of radioimmunoscintigraphy in colorectal cancer]. / Ensayo piloto con el anticuerpo monoclonal ior-C5 como agente potencial de la inmunocentelleografía del cáncer colorrectal.

UNLABELLED: IOR C-5 is a G1 immunoglobulin type intact murine monoclonal antibody (MAb) that was developed in the Center of Molecular Immunology in Havana City, Cuba. In immunohistochemical studies, this demonstrated a significant affinity for the epithelial tissues so that it was used in a pilot clinical study to perform a radioimmunoscintigraphy of the colorectal primary tumors and their locoregional recurrences. It was labeled with 99mTc using the Schwarz method, with a > 95% performance. Planar images of the chest, abdomen and pelvis were performed at 10 minutes, 4-6 hours and 18-24 hours post-injection in the anterior and posterior projections and the SPECT was performed 4-6 hours and 18-24 hours post-injection of 1.85 GBq 99mTC. This study has aimed to verify in vivo the capacity of ior-C5 MAb to accumulate in the malignant colorectal lesions. ior-C5 accumulated in 5 out of the 7 patients who were studied and who were suffering from colorectal cancer or in whom there was suspicion of recurrence. There was a negative case of primary tumors, which was an adenocarcinoma in situ in a tubular-papillary adenoma. The second case with a negative radioimmunoscintigraphy was a true negative case. CONCLUSIONS: It can be concluded that even though the number of patients is quite low, ior-C5 fulfilled the expectations of recognizing the epitope expressed in colorectal tumors in an in vivo human environment.