Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein.

Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.

Non-Hodgkin's lymphomas in Mexico. A clinicopathological and molecular analysis.

It is now well established that the clinical and histopathological characteristics of non-Hodgkin's lymphomas may vary significantly throughout the world. However, only a few reports have been published in Latin American countries. In this work, the clinical and pathologic findings of 264 patients with non-Hodgkin's lymphomas in Mexico City were analyzed. Diffuse large (14%) and diffuse mixed cell types (20%) predominated among nodal lymphomas. Within the group of patients with high grade malignancies, immunoblastic sarcomas were the most common (40/48). It is important to mention that follicular lymphomas were sporadic (4.5%). Among extranodal lymphomas the most commonly involved site was the gastrointestinal tract (11.3%), followed by the midline (6%). Molecular analysis of 65 cases with immunoglobulin and T-cell receptor gene probes showed that most lymphomas were of B-cell lineage (66%). The remaining group was composed of T-cell (25%) and bigenotypic malignancies (9%). All attempts to establish a correlation between the clinical stage and histopathological types with the genetic findings were not successful. However, pre-B and bigenotypic lymphomas were observed mainly in patients over 60 years of age. This study highlights some relevant characteristics of non-Hodgkin's lymphomas in Mexico.

Primary B cell lymphoma of the rectum in a patient coinfected with HIV-1 and HTLV-I.

This report describes a clinical case of a large cell, immunoblastic plasmacytoid malignant B-cell lymphoma of the rectum in an AIDS patient coinfected with HTLV-I. The malignant cells showed clonal genetic rearrangement of the HC (JH) and LCK genes. Infection by EBV was demonstrated serologically and with slot blots using genomic DNA of the cancer cells. Southern blot analysis with DNA extracted from the lymphoma cells were negative for HTLV-I. The patient received seven cycles of VACO-B which induced complete but transient clinical remission of the tumor. The final outcome of the patient is unknown.

Damage in B2m genes and DNA methylation of H-2 genes are involved in loss of expression of class I MHC products on the membrane of LR.4, a cell line derivative of the T-cell lymphoma L5178Y.

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.

Clinical, histologic and genetic characterization of non-Hodgkin's lymphomas in Mexicans.

We describe the anatomical distribution, histological and molecular characteristics of 32 cases of NHL. Staging of the NHL was made according to conventionally accepted schemes. Histologically the NHL were classified in grades following the criteria defined by the Working Formulation. Rearrangements in one or more Ig or TcR receptor genes were detected in Southern blots and allowed us to determine the cell type and stage of differentiation. Serological analysis of 26 serum samples revealed the existence of antibodies against EBV epitopes; eight of these patients carried viral sequences in the tumor genome as determined by slot blot hybridization. Our studies indicate that the use of various methods is of paramount importance in order to improve our understanding of the natural history of NHL.

Immunologic and genetic characterization of S180, a cell line of murine origin capable of growing in different inbred strains of mice.

Some of the immunologic and genetic properties of the cell line S180 have been examined. These cells grew without restrictions in the peritoneal cavity of different inbred strains of mice and invariably killed the animals. With Northern blots it was demonstrated that S180 cells contained class I mRNAs but failed to transcribe B2m genes. However, under experimental conditions, a protective humoral immune response mediated by cytotoxic antibodies and complement against S180 cells was obtained through non-H-2 antigens in C57BL/6J mice.

Molecular karyotype of Entamoeba histolytica and Entamoeba invadens.

We report the size-fractionation of Entamoeba histolytica and E. invadens deoxyribonucleic acid (DNA) by pulsed field gradient electrophoresis. Using 3 different electrophoretic conditions, we were able to resolve 6 to 9 bands between 300 and 2000 kilobases (kb), distributed in 16-22 large and up to 31 small chromosomes for E. histolytica DNA. For E. invadens, 4 to 5 bands between 300 and over 2000 kb were resolved and discriminated in 4 large and 6 small chromosomes. A ribosomal probe from Trypanosoma brucei hybridized with a 1100 kb band in E. histolytica strain HM1:IMSS and with a 1000 kb band in clone A of strain HM1:IMSS. Both cell lines also showed hybridization at the origin. The ribosomal probe hybridized only at the origin of the E. invadens DNA lane. A triosephosphate isomerase DNA probe from T. brucei hybridized only with a 2000 kb band in E. invadens, indicating that banding patterns are chromosomal bands and ruling out the possibility of DNA degradation.

[Identification of Entamoeba histolytica chromosomes by pulsed field gradient electrophoresis]. / Identificación de cromosomas de Entamoeba histolytica por electroforesis en pulsos de campos en gradiente.

The use of pulsed field gradient electrophoresis (PFG) has simplified the study of chromosomic organization in protozoan parasites whose chromosomes do not condense during division. In this paper we used PCG to analyzed chromosome organization Entamoeba histolytica's. In the conditions which we used (200 volts, 24 hours, 3 minute pulses and the anode at 7.5 cm. from the end of the cathode) 6 and 9 band of amebic DNA were resolved. By means of a ribosomal DNA probe from Trypanosoma brucei amebic DNA was detected in the gels representing chromosomal sizes, in accord with the reproducibility of our results and the hybridization pattern we obtained. The numbers of 6 to 9 obtained for PFG is in accord with the results which we have previously reported. The suggest that Entamoeba histolytica has between 12 and 16 structures similar to chromosomes, if Entamoeba histolytica were diploid, and therefore each band would consist of two chromosomes. Nevertheless small condensations of DNA similar to minichromosomes of kinetoplasas cannot be ruled out, and this would considerably increase the number of chromosomes estimated in this paper.

Occurrence of human papillomavirus type 16 DNA sequences and c-myc oncogene alterations in uterine-cervix carcinoma.

Using genetic engineering and molecular biology techniques, we have examined sixteen human carcinomas in the uterine-cervix tumors (the most frequent tumor in México, representing 34% of malignant tumors in women), for the presence of Human Papillomavirus type 16 (HPV-16) DNA sequences and possible alterations of the cellular myc (c-myc) proto-oncogene. In this study we have analyzed cervical carcinomas from patient with clinical stage II. We detected in 31% of these samples, the presence of HPV-16 sequences (2-100 copies). In addition, an elevated amplification (up to 80-fold in one tumor) and/or rearrangement of the c-myc oncogene was detected in most tumors (more than 90% of the samples). These results suggest that either c-myc oncogene and/or HPV-16 could play an important role in the development of uterine-cervix carcinoma.

Novel combination of c-myc, N-myc and N-ras oncogene alteration in brain tumors.

We have examined forty human brain tumors (neoplasias presenting an important incidence in Mexico), for cellular myc (c-myc), N-myc and N-ras proto-oncogene alterations. An elevated amplification and/or rearrangement of the oncogenes was detected in most samples (60% presenting alteration for c-myc, 54% for N-myc, 6% for N-ras and 60% for ras-related genes). The tumors were of different histological types and for some of them we detected either amplification and/or rearrangement of the oncogenes. We describe, for the first time, the alterations of two related genes (c-myc and N-myc) in the same tumor samples; in 64% of the analyzed samples, oncogene alterations were accompanied by enhanced expression of N-myc and ras-related genes. These results suggest an important role for c-myc, N-myc and N-ras oncogenes, in the development and progression of brain tumors.

High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix.

We have examined 35 human tumors of the uterine cervix (carcinoma presenting the highest incidence in Mexico; about 34% of women's malignant tumors) for alterations of the cellular myc (c-myc) protooncogene. Elevated amplification and/or rearrangement of the c-myc oncogene were detected in most (approximately 90%) samples (48% showed amplification and 43% presented both alterations). Most tumors were stage II cervical carcinomas and for some of them we detected up to 60-fold amplification of c-myc. These results suggest an important role for c-myc oncogene in the development of tumors of the uterine cervix.