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The extracellular matrix in skeletal tissue besides being responsible for its structure and function also provides key regulatory signals for cell proliferation and differentiation by interacting directly with cell receptors, controlling the diffusion of soluble growth factors. Immature bone marrow cells can migrate through capillaries to systemic circulation as well as return to bone marrow. This phenomenon is the biological basis for bone marrow transplant. The mechanisms that control the migration and the return of these immature bone marrow cells are still to be described in a generally accepted model. Thirty days-old Wistar rats were employed as recipients for the implantation of ceramic blocks or demineralized femur diaphysis. The material was implanted subcutaneously at the dorsum. The rats were euthanized with CO2 at 15, 40 and 68 days after implantation. The whole area of implantation was harvested from skin deep up to the muscle. Liver, spleen, lungs, femurs and the vertebral spine, ribs and sternum were also harvested. The harvested material was fixed in Millonig formalin and decalcified with EDTA, if applicable. The material was then progressively dehydrated in alcohol, immersed in xylol, impregnated and embedded in paraffin. The paraffin blocks were then sliced in 5µm sections with a microtome, stained in Haematoxilin – Eosin, Lennert's Giemsa and Gomori's reticulin for histological analysis under brightfield optical microscopy. All implants produced an intense fibroblastic reaction, angiogenesis and subsequent modulation into a loose stroma. However, hematopoesis was not observed. Implantation of endogenous haematopoetic cells in an inductive stroma, which might propitiate hematopoesis is a model scarcely studied. Experiments such as the one described here might be good models for understanding the sequence of events leading to formation of the stroma and implantation and differentiation of haematopoetic precursors.

Matrix‐assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI‐MS/MS ion search (using MASCOT) have become the preferred methods for high‐throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post‐source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI‐TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N‐terminal derivatization of the tryptic peptides with 4‐sulfophenyl isothiocyanate (SPITC) prior to MALDI‐TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI‐TOF/TOF‐PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules. Copyright © 2007 John Wiley & Sons, Ltd.

Matrix‐assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI‐MS/MS ion search (using MASCOT) have become the preferred methods for high‐throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post‐source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI‐TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N‐terminal derivatization of the tryptic peptides with 4‐sulfophenyl isothiocyanate (SPITC) prior to MALDI‐TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI‐TOF/TOF‐PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules. Copyright © 2007 John Wiley & Sons, Ltd.

Abstract: To compare the sequential evolution of lesions developed by resistant (A/Sn) and susceptible (B10.A) mice to Paracoccidioides brasiliensis infection we inoculated a virulent isolate of the fungus and collected the pancreas/peripancreatic omentum monthly (from 1 to 6 months) post infection. After fixation, tissue sections were stained by conventional methods for light microscopy to investigate the cellular composition, the extracellular matrix (ECM) patterns and the morphology of the yeasts in the lesions. In both strains, the fungal lesions were localized mostly in the omentum; a few lesions in the pancreatic parenchyma were observed, mostly in B10.A mice. In both strains, macrophages and plasmocytes were the predominant cells in all lesions, followed by neutrophils (PMN) and macrophages transformed into giant and epithelioid cells. Remarkable differences were observed between resistant and susceptible mice, specially related to the ECM structure of the granulomatous lesions. In A/Sn mice, from the 1st month on, the coexistence of two types of lesions was observed: one type showed a well-defined encapsulated nodule, constituted mainly of type I collagen. Neutrophils were abundant in areas of massive fungal destruction and few viable yeasts were observed. The other type showed residual characteristics, with sparse collagen deposits and presence of xantomatous-like macrophages, containing degenerated fungi. Such residual lesions predominated after the 2nd month and were the only type observed from the 4th month on, indicating the control of the infection. In B10.A mice, on the contrary, only one type of lesion was observed, showing less tendency to encapsulation and the formation of multiple small granulomatous foci, individualized by reticular type III collagen fibers. There were many plasmocytes in the periphery and large numbers of budding yeasts, with no evidence of fungal destruction. In the course of the infection the lesions progressively increased in number and size. Altogether, the comparative histopathological analysis demonstrates the influence of the genetic pattern of the host on the lesions developed by resistant and susceptible mice to P. brasiliensis infection.

We present a comparative histopathological study of both acute and chronic human adenovirus pneumonia, with reference to the cellular and extracellular matrix components. Seventeen lungs from autopsied patients whose ages ranged from 2 to 60 months were studied. Adenovirus types 1, 2, 3, 5, and 7 were isolated from 15 patients with acute lung disease, and types 2 and 7 were isolated from the other two patients with chronic pulmonary illness. The results indicated the occurrence of two basic patterns of adenovirus interstitial pneumonia: (1) classic pattern (acute), characterized by necrosis and degeneration and many type II pneumocytes with intranuclear inclusion bodies, which were positive for adenovirus DNA by in situ hybridization, and (2) proliferative or proliferative-productive pattern (chronic), which presented with diffuse pulmonary fibrosis and the interstitial proliferation of fibroblast-like cells, compatible with myofibroblasts (positive for vimentin and α smooth muscle actin), and increase in collagen types I and III, elastic fibers, and proteoglycans. Alveolar collapse appears to be an important pathogenetic mechanism in the development of this pattern.

Trypanosoma cruzi is a heterogeneous population of parasites as shown by differences between strains and cloned stock from the same strain. Herein we present evidence of the noninfectivity of CL-14, a clone derived from the CL strain ofT. cruzi. In a previous paper we reported the absence of parasitemia and mortality in mice injected with metacyclic trypomastigotes of this clone. To investigate further this lack of infectivity we did and extensive histopathological analysis in mice at different intervals after i.p. (5 and 15 days as well as 1, 4, and 12 months) or i.v. (5 and 30 days) injection of trypomastigotes. In spite of a systematic search in all tissues and organs of the animals, no parasite or significant pathological change was detected in any of the tissue sections. These data suggest the inability of this clone to mediate infection and/or cause pathological alterations in vivo.

LENZI, H. L., OLIVEIRA, D. N., LIMA, M. T., and GATTASS, C. R. 1996.Trypanosoma cruzi:Paninfectivity of CL strain during murine acute infection.Experimental Parasitology84,16–27. A systematic study of the distribution of intracellular parasites in the organs and tissues of mice acutely infected (15 days) with the CL strain ofTrypanosoma cruziwas performed. Almost all tissues and organs were parasitized with different intensities, including several epithelial cell types. In addition to striated, cardiac, and smooth muscles a very high parasitism of fat cells, pancreas, and genital adnexa was observed. A smaller number of parasites was found in all other structures studied except in highly vascularized structures such as in the penile corpora cavernosa, pulmonary and renal parenchyma, islets of Langerhans, hepatic sinusoids, and in atrial endothelium. This paper also shows, for the first time in the literature, the parasitism of milky spots, cornea epithelium, cornea stroma, retroorbital fibroblasts, seminal vesicles, and coagulative, Cowper's, urethral, preputial, sebaceous anal, and clitoris glands. The results indicated that CL strain is highly invasive, being able to infect cells derived from the three embryonic layers (ectoderm, mesoderm, and endoderm), suggesting that the paninfectivity may influence the outcome of immunological and pathological events.

The oxidative burst of polymorphonuclear neutrophils (PMN) and their ability to inhibit Paracoccidioides brasiliensisgrowth was studied in susceptible (B10.A) and resistant (A/J) mice. The cells were obtained after subcutaneous inoculation in air pouches, yielding highly pure PMN preparations; the number of cells was similar for both strains at 24 h and five times higher in the resistant strain at 15 days. The oxidative metabolism of these PMN was evaluated by the luminol and lucigen‐enhanced chemiluminescence upon stimulation with PMA or killed P.brasiliensis (Pb). At 24 h of infection PMN from both strains showed similar responses. However, at 15 days a great enhancement of the Pb‐stimulated luminol‐enhanced chemiluminescence was observed only in PMN from resistant mice. Such increase was markedly inhibited by the addition of catalase. Independent of the mouse strain or time of infection the lucigenin‐enhanced chemiluminescence showed the same intensity. The lucigenin‐enhanced chemiluminescence of PMN without stimuli from resistant mice did not change with the time of infection, however, after 15 days of infection a significantly lower chemiluminescence was detected with PMN from susceptible mice. At 15 days of infection the PMN from B10.A were unable to kill P. brasiliensisyeast cells in vitro. Because the lucigenin‐ and luminol‐enhanced chemiluminescence detects, respectively, the O2‐production and the myeloperoxidase/hydrogen peroxide halide system, the present data show parallels between deficiency in the production of oxygen‐reactive species by PMN and lower fungicidal activity.