Factors affecting visibility of a target tissue in histologic sections.

The objective of histologic techniques is to stain the subject with high specificity and high visibility. Visibility depends on the microscope's resolution and contrast and on the microscopist's skill at optimizing the microscope's image. It also depends on histotechnological factors, which include specificity and differentiation of the stain, density of background staining (particularly in silver stains), innate color, and grayscale contrasts of the dyes in the stains and color and density of the counterstain. If contrast is not optimal, the image should be evaluated on the basis of 2 types of contrast-color and grayscale. Complementary colors have maximum color contrast, and the color triangle is useful in the selection of a suitable counterstain. Grayscale contrast is a function of the density of a stain. If dyes capable of staining the target and backgrounds tissue do not have optimal color contrast, the only method of increasing contrast is to change the grayscale value of one of the stains, usually the counterstain. Colors can have a subconscious effect on a viewer. Depending on whether they are aesthetically pleasing, they may influence the rigor of and time spent on the histopathologic examination. Maximizing the specificity of stains such as hematoxylin, eosin, trichrome, and Luxol fast blue (LFB) depends on optimal differentiation. In differentiation of counterstains such as methylene blue in the Ziehl-Neelsen stain, its recommended density is conveniently expressed as a grayscale value. Independent evaluation of color and grayscale contrasts is very helpful in determining the cause of low contrast in an image. This review discusses aspects of the histotechnique affecting the visibility of tissue components.

Axonal degeneration in sheep caused by the ingestion of Halimium brasiliense.

Nervous system disease is reported in sheep from 2 farms in southern Brazil and in 33 farms in Uruguay. The illness was seasonal, occurring from May to November, during the growing season of Halimium brasiliense, and primarily affected sheep older than 3 years of age. Clinical signs included transient seizures that occurred mainly when sheep were disturbed or frightened. Most affected sheep recovered when removed to other pastures. Feeding trials produced clinical signs in 1 sheep after the ingestion of 2,117 g/kg of body weight of H. brasiliense over 142 days. Two sheep that had previously recovered from spontaneous toxicosis developed clinical signs after the ingestion of 263 g and 565 g of H. brasiliense per kg body weight given over 36 and 31 days, respectively. The main histologic lesion was vacuolation of the brain and spinal cord, with rare axonal spheroid formation. Transmission electron microscopy revealed segmental axonal swelling with degeneration and disappearance of the axonal organelles and vacuolation of the axoplasm. A pigment identified as ceroid was also present in neurons, astrocytes, and macrophages. These lesions suggested a novel morphologic manifestation of a toxic axonopathy.

The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein (WASp)-mediated actin polymerization to T cell antigen receptor endocytosis.

Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (DeltaDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

Prevalence and mechanisms of macrolide resistance in invasive and noninvasive group B streptococcus isolates from Ontario, Canada.

Macrolide resistance has been demonstrated in group B streptococcus (GBS), but there is limited information regarding mechanisms of resistance and their prevalence. We determined these in GBS obtained from neonatal blood cultures and vaginal swabs from pregnant women. Of 178 isolates from cases of neonatal GBS sepsis collected from 1995 to 1998, 8 and 4.5% were resistant to erythromycin and clindamycin, respectively, and one isolate showed intermediate penicillin resistance (MIC, 0.25 microg/ml). Of 101 consecutive vaginal or rectal/vaginal isolates collected in 1999, 18 and 8% were resistant to erythromycin and clindamycin, respectively. Tetracycline resistance was high (>80%) among both groups of isolates. Of 32 erythromycin-resistant isolates, 28 possessed the erm methylase gene (7 ermB and 21 ermTR/ermA) and 4 harbored the mefA gene; one isolate harbored both genes. One isolate which was susceptible to erythromycin but resistant to clindamycin (MIC, 4 microg/ml) was found to have the linB gene, previously identified only in Enterococcus faecium. The mreA gene was found in all the erythromycin-resistant strains as well as in 10 erythromycin-susceptible strains. The rate of erythromycin resistance increased from 5% in 1995-96 to 13% in 1998-99, which coincided with an increase in macrolide usage during that time.

Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.

Variance in fibronectin binding and fnb locus polymorphisms in Staphylococcus aureus: identification of antigenic variation in a fibronectin binding protein adhesin of the epidemic CMRSA-1 strain of methicillin-resistant S. aureus.

The fnbA and fnbB genes of Staphylococcus aureus 8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of fnb genes or the number of D motifs, we identified five polymorphism groups. S. aureus 8325-4 and 91% of methicillin-resistant S. aureus (MRSA) isolates belong to polymorphism group I, with two fnb genes and three copies of the D motif. Polymorphism group II contained one fnb gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number of fnb genes or the number of D motifs. Therefore, the fnb locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.

Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease.

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Molecular analysis of the accessory gene regulator (agr) locus and balance of virulence factor expression in epidemic methicillin-resistant Staphylococcus aureus.

Potential relationships between virulence factor expression and transmissibility were assessed in epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones CMRSA-1 and CMRSA-3. A major subtype of CMRSA-1 exhibited normal transcription of RNAIII, which facilitates the induction of secreted virulence factors and repression of colonization factor expression at high cell density. However, these isolates characteristically did not express alpha-toxin or protease and displayed a limited profile of secreted proteins. CMRSA-1 also expressed a novel cell surface glycoprotein and exhibited a unique polymorphism within the accessory gene regulator (agr) locus. CMRSA-3 displayed attenuated activation of RNAIII transcription, which was consistent with its higher fibronectin-binding and coagulase activity relative to sporadic MRSA or CMRSA-1 (P=.05), low protease activity, and limited profile of secreted proteins. Thus, the balance of virulence factor expression in CMRSA-1 and CMRSA-3 favors the colonization phase of infection, and CMRSA-1 possesses unique genotypic and phenotypic traits.

Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essential for fibronectin binding.

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1(21-34) and D3(20-33), which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3(20-33) immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes.

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele (WAS(-)(/)(-)). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS(-)(/)(-) lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS(-)(/)(-) neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.

Transgenic mouse model of AA amyloidosis.

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

Magnetic resonance imaging appearance of intracranial hemorrhage secondary to cerebral vascular malformation in a dog.

A 14-year-old dog developed an acute onset of depression, disorientation, left hemiparesis,left hemianopia, left facial hypoesthesia, and a tendency to turn to the right. Based on these findings, a lesion affecting the right forebrain was suspected. Magnetic resonance imaging showed a mass within the right cerebral hemisphere resulting in compression of the right lateral ventricle and shifting the longitudinal fissure to the left. The lesion was hyperintense on T1-weighted images and hyperintense with focal regions of hypointensity on proton density-, and T2-weighted images, consistent with a subacute hemorrhage. At necropsy, there was a hematoma in the parietal portion of the right cerebral hemisphere. The hemorrhage was surrounded by numerous thin-walled veins, most likely a venous malformation. Magnetic resonance imaging of intracranial hemorrhage is reviewed.

Bilateral renal metastases of nasal chondrosarcoma in a dog.

Chondrosarcoma is the most common nonepithelial sinonasal neoplasm in the dog, and metastasis is considered rare. A 7-year-old Irish Setter had bilateral renal enlargement 17 months following surgery and radiotherapy for a primary nasal chondrosarcoma. Histologic evaluation revealed chondrosarcoma metastases in both kidneys. A diagnosis of nasal chondrosarcoma with bilateral renal metastasis was made. The clinical importance of this report is that routine recommendations for the evaluation of regional and/or distant metatasis in a dog with a dignosis of nasal chondrosarcoma, namely routine whole body physical examination and thoracic radiography, failed to demonstrate the presence of abdominal metatases, which ultimately led to the demise of this dog. The biologically aggressive nature of this chondrosarcoma of the nasal cavity indicates that additional information is needed before a prognosis can be reliably established for dogs with this tumor type.

Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease.

The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity.

Parosteal osteosarcoma of the cervical vertebra in a dog.

A 12-year-old Maltese terrier was evaluated for progressive tetraparesis and neck pain. On radiographs, there was a periosteal reaction involving the fourth cervical vertebra. Myelographically, there was extradural compression of the spinal cord associated with the lesion. The dog was euthanized and necropsied. Histopathologic diagnosis was parosteal osteosarcoma of the vertebra.

Identification of D motif epitopes in Staphylococcus aureus fibronectin-binding protein for the production of antibody inhibitors of fibronectin binding.

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.