LAT (linker for activation of T cells): a useful marker for megakaryocyte evaluation on bone marrow biopsies

Detection of atypical megakaryocytes in bone marrow biopsies, especially in cases of myelodysplastic syndromes (MDS), chronic myeloproliferative disorders (CMPD) and acute leukemias, is facilitated by staining for markers such as Ulex europaeus agglutinin (UEA)-J, CD31, CD61 and von Willebrand factor (VWF), the latter being considered the most sensitive. Recently, LAT (linker for activation of T cells), a molecule involved in T-cell activation and platelet aggregation, was found to be expressed by megakaryocytes and platelets in tissue sections. We compared VWF and LAT immunoreactivity on megakaryocytes in 64 bone marrow biopsies from 12 normal controls (NC), and from patients with MDS (n=18), CMPD (n=21) and acute megakaryocytic leukemia (AML-M7, n=13). Immunostaining was performed on paraffin sections with polyclonal antibodies against VWF and LAT. Immunoreactivity was evaluated by counting positive megakaryocytes in 10 high-power fields, and values were compared using Student's t test for paired data. Both VWF and LAT predominantly stained the cytoplasm of megakaryocytes, although LAT was also recognizable on the cell membrane. In most biopsies, the immunoreactivity of the two antibodies was quite similar. No significant differences were noticed between the mean values of VWF+ and LAT+ megakaryocytes. However, in 22 cases (5 NC; 5 MDS; 6 CMPD; 6 AML-M7), the number of LAT+ megakaryocytes was at least 30% higher than VWF+cells, while in 3 cases opposite findings were found. In 3 AML-M7 cases, anti-LAT antibodies stained numerous megakaryocytes, but anti-VWF staining was practically negative; in another 5 AML-M7 cases, anti-LAT labeling was much stronger than anti-VWF staining. LAT represents a useful immunohistochemical marker for megakaryocytes in normal and pathological conditions. It seems to be expressed by megakaryocytes more than VWF in most cases and, particularly, in conditions associated with poorly differentiated megakaryocytes, such as acute megakaryocytic leukemias. The use of LAT staining should be recommended in association with other megakaryocyte markers in the study of bone marrow biopsies in cases of hematopoietic disorders.     

Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.     

Substance P (neurokinin-1) and neurokinin A (neurokinin-2) receptor gene and protein expression in the healthy and inflamed human intestine

Increasing evidence suggests that tachykinins are involved in the control of pathophysiological states, such as inflammation. The precise localization of tachykinin receptors is of paramount importance in the search for their possible physiological and pathological role; in this study, therefore, we attempted to define cellular sites of substance P (NK-1R) and neurokinin A (NK-2R) receptor expression in the healthy and the inflamed human intestine by in situ hybridization and immunohistochemistry. In the normal ileum and colon, NK-1R and NK-2R were localized to smooth muscle cells of the muscularis mucosae and propria and a few inflammatory cells of the lamina propria; NK-1R expression was also found in the muscular wall of submucosal blood vessels, enteric neurons and, to a lesser degree, in surface epithelial cells. Patients with Crohn's disease and ulcerative colitis showed a dramatic increase in NK-1R density relative to controls, in both the inflamed and the uninvolved mucosa. Up-regulation of NK-1R was particularly evident on epithelial cells lining the mucosal surface and crypts, as well as on endothelial cells of capillaries and venules. Also, a marked increase in NK-2R expression was found in both groups of patients on inflammatory cells of the lamina propria, especially eosinophils. Our findings demonstrate that in the normal human intestine NK-1R and NK-2R are expressed in multiple cell types, which are endowed with different physiological functions; in addition, they demonstrate that both NK-1R and NK-2R are up-regulated in patients with Crohn's disease and ulcerative colitis. Taken together, these observations may have important physiological and pathophysiological implications, and provide the rationale for the use of NK-1R and NK-2R antagonists in the treatment of inflammatory bowel disease.     

Six-month multicenter study on invasive infections due to Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis in Argentina

During a 6-month period, 95 invasive infections due to Streptococcus pyogenes and group C or group G Streptococcus dysgalactiae subsp. equisimilis were recorded from 40 centers of 16 cities in Argentina. We describe here epidemiologic data available for 55 and 19 patients, respectively, associated with invasive infections due to S. pyogenes and S. dysgalactiae subsp. equisimilis. The associated isolates and 58 additional pharyngeal isolates were genotyped and subjected to serologic and/or antibiotic susceptibility testing. Group A streptococcal emm type distribution and strain association with toxic shock appeared to differ somewhat from results found within the United States; however, serologic characterization and sof sequence typing suggested that emm types found in both countries are reflective of shared clonal types.     

Prevalences, genotypes, and risk factors for HIV transmission in South America

HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.     

Semantic relatedness effects in conjoined noun phrase production: implications for the role of short-term memory

Speech error data and empirical studies suggest that the scope of planning is larger for semantic than for phonological form representations in speech production. Previous results have demonstrated that some patients show dissociable impairments in the retention of semantic and phonological codes. The effect of these STM deficits on speech production was investigated using a phrase production paradigm that manipulated the semantic relatedness of the words in the phrase. Subjects produced a conjoined noun phrase to describe two pictures (e.g., "ball and hat") or produced the same phrases in response to pairs of written words. For the picture naming condition, control subjects showed an interference effect for semantically related pictures relative to unrelated pictures. This interference effect was greatly exaggerated for two patients with semantic short-term memory deficits but not for a patient with a phonological STM deficit. For the written words, control subjects showed a small facilitatory effect for the onset of phrases containing semantically related words. One of the patients with a semantic STM deficit who was tested on picture naming was also tested on these materials and showed a small facilitatory effect within the range of controls. The findings support the contention that speech planning is carried out at a phrasal level at the lexical-semantic level and that the capacities that support semantic retention in list recall support speech production planning.     

Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis.

Expression of platelet-derived growth factor (PDGF) and its receptor (R) subunits was evaluated in normal human liver and in cirrhotic liver tissue by in situ hybridization and immunohistochemistry. In normal liver, PDGF and PDGF-R subunit expression was limited to a few mesenchymal cells of the portal tract stroma and vessels. In cirrhotic liver, PDGF-A and -B chain mRNA expression was markedly increased and was co-distributed with immunoreactivity for PDGF-AA and -BB in infiltrating inflammatory cells and along vascular structures within fibrous septa. These aspects were paralleled by a marked overexpression of PDGF-R alpha- and beta-subunit mRNAs and of the relative immunoreactivities in a wide range of mesenchymal cells in fibrous septa and in perisinusoidal alpha-smooth-muscle-actin-positive cells. In general expression and distribution of PDGF-R subunits appeared to be related to the activation of different mesenchymal cell types involved in the fibroproliferative process. Therefore, we evaluated the expression of PDGF-R subunits in liver tissue specimens with increasing degrees of necroinflammatory activity. The results of this additional study confirmed that expression of PDGF-R subunits is highly correlated with the severity of histological lesions and collagen deposition. Our results, providing evidence for a functional involvement of PDGF/PDGF-R in liver fibrogenesis, greatly support the results of previous in vitro studies and direct attention toward pharmacological strategies able to affect the series of signaling events arising from the autophosphorylation of PDGF-R subunits.     

Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis. The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.