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Molecular profiling of tissue samples using laser capture microdissection.

The management of cancer and other genetically based diseases is far from optimal in even our most advanced medical centers. There is still uncertainty regarding how diseases will progress in certain patients, toxicity that must be tolerated with imprecise treatment regimens and significant potential for treatment failure. As our understanding of the complexity of these diseases has increased, it has become clear that we must move toward precisely tailored approaches to treating each individual patient. To that end, a major goal of current medical research is the rapid identification of the specific molecular alterations in each patient's disease. This will enable the design of optimal diagnosis, prognosis and treatment, significantly improving survival. This review describes one important approach to the genetic analysis of disease--molecular profiling--and the tenets and technologies necessary for its success.

DNA Fingerprinting↗

[Insecticide poisoning. Microdissection of nerves and muscle histochemistry in 10 cases].

A sural nerve and a muscle biopsy study of patients with chronic insecticides poisoning, with teased fiber preparations, routine pathologic studies of nerves and histochemistry of muscle is reported. The sural nerves of ten patients were studied and a teased fiber preparation was done in nine. The tenth patient had only fibrosis and no myelin was found. The sural nerves were abnormal in all patients and the teased fiber preparation resulted in preponderance of type C, D and large amount of G type fibers, according to Dyck's classification. These fibers had enlargement of the axon and myelin sheath, also seen in routine sections. The muscle biopsy with routine and histochemistry methods was done in 8 cases; in 6 there was found signs of denervation; the remaining cases were normal, but these were proximal muscles. The authors conclude that the process primarily interfere with the functions of the axons, with distal axonal degeneration and a dying back phenomen.

Adult↗

[Familial spastic paraplegia with amyotrophy. Clinical, electromyographic, histochemical study and microdissection].

Four cases of familial spastic paraplegia with amyotrophy in siblings from a consanguineous married are reported. The routine laboratory examination were normal. The electromiography and muscle biopsy processed by histochemistry showed signs of denervation with reinervation. The motor nerve conduction velocity was decreased in the peroneal nerve in 3 cases. The teased fiber preparation of sural nerves was abnormal in four cases. It was found increased of C, D and G fibers suggesting demyelination with secondary remyelination. The authors believe the abnormalities found could be due the distal axonal degeneration, with secondary regeneration and suggest the hypothesis that the fact is an axoplasmic flow defect in the central and peripheral nervous system.

Adult↗

Modified tannin-osmium conductive staining method for non-coated scanning electron microscope specimens. Its application to microdissection scanning electron microscopy of the spleen.

Supplement of guanidine hydrochloride to the original tannic acid solution of MURAKAMI (1973) eliminated, without debilitating the conductivity effect, the vexatious treatment with different amino-acids and sucrose in the previous method (MURAKAMI, 1974). The procedures and advantages of this simplified modification are described using human spleen samples; attempt was made to elucidate the structure of arterial terminals by dissecting the specimens under the scanning electron microscope.

Dissection↗

Identification of receptor ligands by screening phage-display peptide libraries ex vivo on microdissected kidney tubules.

A novel method to identify receptor ligands for defined renal tubular segments has been developed. Ex vivo screening of phage-display peptide libraries on isolated intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD) allowed the direct access of phage to the basolateral surface of tubular epithelial cells. Two distinct peptide motifs were selected for CCD and PCT, indicating differential expression of some membrane receptors on the basolateral surface of defined kidney tubule segments. Using the linear peptide motif ELRGD(R/M)AX(W/L), recovered from freshly isolated rat CCD, mediated 16-fold selectivity of phage binding to CCD compared with PCT. Binding to CCD was 39-fold higher than that of a random control phage. Binding and subsequent internalization of phage, most likely by an integrin-mediated endocytosis pathway, was abolished by the addition of the corresponding synthetic peptide. Furthermore, the results demonstrate that presentation and flanking amino acids determine the specific binding properties of RGD ligands to their putative integrin receptors. The results emphasize the need of a native cell system for the identification of renal epithelial cell surface ligands. Such ligands are of potential relevance for the analysis of interactions between extracellular matrix and kidney tubules or for the development of improved vectors for kidney-specific drug delivery or gene transfer.

Animals↗