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Aims To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. Methods and results Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. Conclusions Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Summary: Increased tolerance of Plasmodium falciparum to front-line artemisinin antimalarials (ARTs) is associated with mutations in Kelch13 (K13), although the precise role of K13 remains unclear. Here, we show that K13 mutations result in decreased expression of this protein, while mislocalization of K13 mimics resistance-conferring mutations, pinpointing partial loss of function of K13 as the relevant molecular event. K13-GFP is associated with ∼170 nm diameter doughnut-shaped structures at the parasite periphery, consistent with the location and dimensions of cytostomes. Moreover, the hemoglobin-peptide profile of ring-stage parasites is reduced when K13 is mislocalized. We developed a pulse-SILAC approach to quantify protein turnover and observe less disruption to protein turnover following ART exposure when K13 is mislocalized. Our findings suggest that K13 regulates digestive vacuole biogenesis and the uptake/degradation of hemoglobin and that ART resistance is mediated by a decrease in heme-dependent drug activation, less proteotoxicity, and increased survival of parasite ring stages. : Artemisinin resistance in P. falciparum is associated with Kelch13 (K13) mutations. Yang et al. report that K13 is located in cytostome-like structures, consistent with a role in hemoglobin uptake. K13 is less abundant in mutants, and hemoglobin catabolism is impaired. The resultant decrease in artemisinin activation likely underpins artemisinin resistance. Keywords: malaria, artemisinin, antimalarial, kelch13, proteomics, SILAC, protein turnover, cytostome

Introduction: \ud Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement.\ud \ud Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,\ud \ud Methods: \ud Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.\ud \ud Results: \ud Immunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).\ud \ud Conclusions: \ud These results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

IntroductionFrozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the stromal compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,MethodsShoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Stromal activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.ResultsImmunohistochemistry demonstrated increased expression of stromal activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).ConclusionsThese results show that stromal fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated stromal fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.

In vertebrates, individual Golgi stacks are joined into a compact ribbon structure; however, the relevance of a ribbon structure has been elusive. Here, we exploit the finding that the membrane tether of the trans-Golgi network, GCC88 (encoded by GCC1), regulates the balance between Golgi mini-stacks and the Golgi ribbon. Loss of Golgi ribbons in stable cells overexpressing GCC88 resulted in compromised mechanistic target of rapamycin (mTOR) signaling and a dramatic increase in LC3-II-positive autophagosomes, whereas RNAi-mediated depletion of GCC88 restored the Golgi ribbon and reduced autophagy. mTOR was absent from dispersed Golgi mini-stacks whereas recruitment of mTOR to lysosomes was unaffected. We show that the Golgi ribbon is a site for localization and activation of mTOR, a process dependent on the ribbon structure. We demonstrate a strict temporal sequence of fragmentation of Golgi ribbon, loss of Golgi mTOR and subsequent increased autophagy. Golgi ribbon fragmentation has been reported in various neurodegenerative diseases and we demonstrate the potential relevance of our findings in neuronal cells using a model of neurodegeneration. Overall, this study highlights a role for the Golgi ribbon in pathways central to cellular homeostasis.This article has an associated First Person interview with the first author of the paper.

Recently, platelet-rich plasma (PRP) has been proven to be an effective regeneration adjunct when combined with autogenous bone in the reconstruction of mandibular defects. However, little is known about the effect of PRP when combined with a bone allograft in the maxillary sinus. The purpose of this study was to quantitatively evaluate the ability of PRP to enhance bone regeneration in the maxillary sinus of sheep when combined with demineralized freeze-dried bone allograft (DFDBA) and cortical cancellous freeze-dried bone allograft (CCFDBA). Ten sheep were selected for bilateral sinus augmentation DFDBA + CCFDBA + PRP (test) and DFDBA + CCFDBA (control). Five were sacrificed at 3 months and the other 5 at 6 months. Hematology tests were performed for platelet count, and histology slides were obtained for histomorphometric analysis taking 2 measures of interest: total area (square millimeters) and percentage of bone fill. Student t tests showed no significant difference between test and control groups for total area (P > .25) and percentage of bone fill (P > .80) at either 3 or 6 months. The control group showed no statistical difference for total area (P < .095) and percentage of bone fill (P < .60) between 3- and 6-month healing times. The test group, however, showed a significant increase in total area (P < .025) but not in percentage of bone fill (P < .40) for the 2 healing periods. When the treatments were compared for interactions within the animal model, no clear tendency was evident for the test group to perform in relation to the control group regarding total area (r = .766, P < .01). A moderate tendency existed between the percentages of bone filled (r = .824, P < .005). Platelet-rich plasma showed higher platelet count than did the whole blood (2 to 5 times). However, no correlation was found between the log ratio and the bone measures. Within the limitations of this study, PRP failed to enhance or accelerate bone regeneration in the maxillary sinus of sheep when combined with bone allograft.

Summary The Plasmodium falciparum genome contains genes encoding three a -ketoacid dehydrogenase multien- zyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chlo- roplasts, respectively, that are part of the organelle- specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum . Protein expression of mitochondrial LipDH and mitochondrial branched chain a -ketoacid dihydrolipoamide transa- cylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N- terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle- specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physi- cochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial a a a - ketoglutarate dehydrogenase and branched chain a a a - ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum .

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

ATP-binding cassette (ABC) transporters serve a variety of physiological functions as well as play key roles in drug resistance. The genome of the human malaria parasite, Plasmodium falciparum, encodes multiple members of this family, one of which, gABCG2, is transcribed predominantly in the gametocyte stage. Here we use gene deletion and tagging to investigate the expression, localization and function of gABCG2. The protein is found in a single dot-like lipid-rich structure within female, but not male, gametocytes. gABCG2-knockout cell lines produce more gametocytes of both sexes. By contrast, cholesteryl esters, diacylglycerols and triacylglycerols are significantly reduced in gABCG2-knockout gametocyte stages. We propose a role for gABCG2 in the regulation of gametocyte numbers and in the accumulation of neutral lipids, which are likely important for parasite development in the insect stages of the parasite life cycle.

Brain cells manufacture and secrete angiogenic peptides after focal cerebral ischemia, but the purpose of this angiogenic response is unknown. Because the maximum possible regional cerebral blood flow is determined by the quantity of microvessels in each unit volume, it is possible that angiogenic peptides are secreted to generate new collateral channels; other possibilities include neuroprotection, recovery/regeneration, and removal of necrotic debris. If the brain attempts to create new collaterals, microvessel density should increase significantly after ischemia. Conversely, if angiogenic-signaling molecules serve some other purpose, microvessel densities may increase slightly or not at all. To clarify, the authors measured microvessel densities with quantitative morphometry. Left middle cerebral arteries of adult male Sprague–Dawley rats were occluded with intraluminal nylon suture for 4 hours followed by 7, 14, 19, or 30 days of reperfusion. Controls received no surgery or suture occlusion. Changes in microvessel density and macrophage numbers were measured by light microscopic morphometry using semiautomated stereologic methods. Microvessel density increased only in the ischemic margin adjacent to areas of pannecrosis and was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascularity changes compared with normal animals. These data suggest that ischemia-induced microvessels are formed to facilitate macrophage infiltration and removal of necrotic brain.

This preclinical rat pilot study quantifies retinal microvessel, endothelial, and pericyte population changes produced by proton irradiationThe left eyes of rats were irradiated with single doses of 8, 14, 20, and 28 Gy protons; right eyes, with two fractions. Animals were euthanized, and eyes were removed; elastase digests were prepared, and cell populations were counted in sample fields. Results were compared with unirradiated controls.Progressive time- and dose-dependent endothelial cell loss occurred following all schedules. Cell loss was significantly different from control values (p0.001) following 28 Gy and following 20 Gy (p0.05) in a single dose. Endothelial cell loss was the same for single- and split-dose schedules. Progressive endothelial cell loss produced vessel collapse and acellular vessel strands. Endothelial cells were in the G(0) phase of the mitotic cycle. 28 Gy produced photoreceptor cell loss.The retinal digest is an elegant bioassay to quantify the microvessel population response. Single- and split-dose schedules appear to yield similar outcomes, in terms of endothelial cell density.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na+, K+-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na+, K+-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.

Periradicular surgeries were performed on the maxillary cuspid teeth of twelve cats. Before reapproximation of the surgical flaps, eight of the osteotomies were covered with a resorbable membrane and eight were filled with human osteogenic protein-1 (hOP-1) on a collagen carrier. The remaining eight sites received no further treatment and served as controls. The animals were euthanized after 12 wk, and the specimens were examined histomorphometrically for the presence or absence of osseous regeneration, inflammation, and cementum formation on the root ends. The results showed that the sites treated with the membrane exhibited significantly more inflammation adjacent to the resected root ends (p0.05), and that the use of the membrane had no statistically significant effect on osseous healing or new cementum formation. The use of hOP-1 was associated with a significant decrease in the thickness of new cementum formed on the resected root ends (p0.05), but had no statistically significant effect on osseous healing or degree of inflammation. Based on these results, it seems that neither the use of hOP-1 nor resorbable membranes have a positive effect on periradicular tissue healing in endodontic surgery.

Problem. The role of bacteria has been well established in pulpal and periapical diseases, but the contribution of the host defenses is less clear. Objectives. The purpose of this study was to compare periradicular lesion development in immunosuppressed rats with that in normal rats. Study Design. Fifteen rats were given weekly injections of Cytoxan (Bristol Laboratories) to suppress their immune systems. The pulps of mandibular first molars of these animals and another 15 rats that had received no medications were exposed and left open to their oral flora. The rats were killed at 2, 4, and 6 weeks. Radiographic analysis was performed by means of a computer linked to a digitizing board and stylus. In addition, specimens were decalcified, sectioned, stained, and examined under a microscope with a grid to quantify relative percentages of surface areas of bone, root, periodontal ligament, marrow spaces, soft tissue, and inflammatory infiltrate. Results. Statistical analysis showed a significantly greater radiographic bone loss in the immunosuppressed group only at 4 weeks. No significant histologic differences were found between the two groups. Conclusion. Our results suggest that reduction of circulating leukocytes may not significantly affect the development of periradicular pathosis in rats.

In the rat, 1 week of a calcium-deficient diet will recruit large numbers of osteoclasts to long bone endosteal surfaces. Subsequent calcium supplementation causes the osteoclasts to disappear in 1-3 days but as little as 3 hours of calcium supplementation reduces the extent of their ruffled borders. To test the hypothesis that at some point there is an irreversible inhibition of osteoclasts, male, weanling, calcium-deficient, SD rats were given various amounts of calcium-containing diet followed by a 12-hour fast. No changes in seven morphological indices of osteoclast activity were found. The hypothesis that the fast had reversed the effects of the calcium diet was supported by a second experiment indicating that no inhibition threshold had been reached. Another experiment showed differences in the degree of osteoclast inhibition with different amounts of calcium supplementation, again without evidence of a threshold. These experiments raised two questions: (1) Will fasting recruit osteoclasts in calcium-replete rats? and (2) Is osteoclast recruitment facilitated by the presence of postosteoclasts? The results of experiments testing these hypotheses support the conclusion that fasting and calcium deficiency maintain plasma calcium levels by different mechanisms, and post-osteoclasts are not available for reactivation. It is concluded that inhibition of osteoclasts by dietary calcium is a graded phenomenon, and when osteoclasts have lost contact with the bone surface they are unavailable for reactivation; a threshold has been reached.

3-D Structured illumination microscopy (SIM), a super-resolution optical microscopy technique that allows an 8-fold increase in volume resolution, is providing new views of the cellular substructure of the malaria parasite, Plasmodium falciparum. © 2011 IEEE.

The pulps of mandibular molars of 15 athymic and 15 conventional rats were surgically exposed and left open to their oral flora. Each group was divided into three subgroups of five animals each. The rats were killed after their pulps were exposed for 2, 4, or 8 weeks. After fixing, decalcifying, and embedding, the specimens were sectioned and stained with hemotoxylin and eosin. They were then examined under a microscopic grid and quantified by percentages of surface areas of bone, connective tissue, bone marrow, intrabony spaces, periapical lesions, and numbers of osteoclasts, with the use of a DataVoice computerized data collection and analysis system. Statistical analysis showed no significant difference between periapical tissue responses of the conventional and athymic groups. The results indicate that the pathogenesis of periapical lesions is a multifactorial phenomenon and is not totally dependent on the presence of T-cell lymphocytes.

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.

Background Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp 60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. Conclusion Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 microm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.

Calcium hydroxide has been the material of choice for apexification. The purpose of this study was to compare the efficacy of osteogenic protein-1 and mineral trioxide aggregate with that of calcium hydroxide in the formation of hard tissue in immature roots of dogs. Sixty-four roots of premolars were used. After induction of periradicular lesions, the canals were debrided and filled with calcium hydroxide for 1 wk. After the removal of calcium hydroxide, the root canals received one of the treatment materials in a balanced design. The animals were euthanized 12 wk later. The degree of hard tissue formation and amount of inflammation were evaluated histomorphically. Data were statistically evaluated using ANOVA, x 2 , and Kruskal-Wallis. Mineral trioxide aggregate produced apical hard tissue formation with significantly greater consistency. The difference in the amount of hard tissue produced among the three test materials was not statistically significant. Furthermore, the degree of inflammation was not significantly different between the various test groups.

To quantify and compare blood flow in maxillary mucogingival flaps, a total of 16 cats (32 mucogingival specimens) were divided into two groups. In group I the blood flow in attached or reflected maxillary tissues were measured. In Group II the effects on blood flow were measured in reflected tissues after the injection of the following solutions: (1) saline solution; (2) 2% lidocaine plain; (3) 2% lidocaine with 1:50,000 epinephrine; (4) no injection. After an injection of 153 Gadolinium microspheres into the left ventricle of the heart, the tissues were harvested and placed in a gamma counter. Group I had a mean blood flow value of 34.4 ml/min/100 gm of tissue for the unreflected samples and 152.3 ml/min/100 gm of reflected tissue. The levels of blood flow in tissue samples in group II results were: no injection (control), 41.2 ml/min/100 gm; saline solution, 46.8 ml/min/110 gm; 2% lidocaine with 1:50,000 epinephrine, 60.6 ml/min/100 gm; and 2% lidocaine plain, 90.2 ml/min/100 gm. On the basis of these results it appears that reflection of gingival tissue and injection of 2% lidocaine plain induces increased blood flow 90 minutes postoperatively.

A method for the estimation of the size and total number of calcitonin-containing cells (C cells) in the rat thyroid gland has been devised. The total area, the number of C cells per unit area, and the areal fraction of C cells were determined for the C cell region using step serial sections. From these data it was estimated that from 0.3 X 10(6) to 1.0 X 10(6) C cells were evenly divided between the two thyroid lobes. Approximately 150 micron3 of cytoplasm were associated with each of these cells. In comparison with sham-operated rats, pinealectomy had little effect on the number of C cells. In an experiment terminated in the summer, there was a statistically insignificant decrease 6 weeks postsurgery; no effect was seen at 12 weeks. On the other hand, a slight increase in the number of C cells was seen in January, 12 weeks postsurgery. The volume of cytoplasm per cell was not altered by pinealectomy.

Both transmission and scanning electron microscopy were used in a study of the ultrastructure of the ultimobranchial gland of the rainbow trout. The gland is a bilateral cystic structure in close relation to the sinus venosus, and the epithelium is composed of two cell types which are interspersed throughout the epithelium. The cells containing secretion granules are columnar and often send microvillous processes out into the lumen. The cell which most often lines the luminal surface, however, is a nongranular supporting cell with short microvilli. The microvilli of the granular cells seem to protrude at the periphery of the dome-shaped supporting cells when viewed from the luminal surface. Characterization of the cellular organelles of these two types of cells was carried out as well as a study of their physical interrelationships.

We have reviewed the morphologic, electrophysiologic, biochemical, and functional methods of evaluating PN regeneration in animal models. There are a large number of anatomic techniques that can provide clear insights into the processes of peripheral nerve regeneration. Since many of these are costly in terms of labor, careful selection of the technique appropriate for the question asked is important. Two of the more important questions are: 1) What are the neurotrophic factors produced by the distal segment that attract the growing axon tip? and 2) What are the components of the basal lamina that facilitate the directed growth of the axons? To answer these questions, whole mount preparations provide the means to economically evaluate the result of experimental manipulation of the environment. Automated nerve fiber counts will be increasingly used to help interpret electrophysiologic studies. Quantitative as well as descriptive ultrastructural analyses will continue to provide valuable data that will be needed in the interpretation of biochemical and histochemical studies. Immunohistochemical probes are sure to become more important as the range of their specificities broadens. With the diversity of anatomic methods available and their capacity to help us visualize the processes occurring during nerve regeneration they will remain a key tool in these studies. Electrophysiologic methods that integrate the CAP and correlate it with the number of functioning NF are most useful. Functional methods are beginning to become more objective and quantitative. The most precise measurements are muscle weight and the isometric response of muscle to tetanic contraction. Sensory function has now been measured objectively by Horch. Single methods of measuring PN regeneration give only limited data, but by combining methods a better understanding of PN regeneration is possible. While understanding the limitations of each method and technique, multi-parameter animal models may provide data most helpful clinically. However, because of great species variability in the reparative response, caution must be given not to extrapolate too much from animal studies. We urge investigators to use the most objective methods available to measure nerve regeneration. Recognizing these limitations, however, animal studies will continue to provide significant insights into PN regeneration and should point the way to improved clinical practice.

Calcitonin-containing cells in serial, 6-micrometer sections of the thyroid glands of Swiss Webster mice, at 1 day, 2 weeks, 4 weeks and 8 weeks of age, were demonstrated by an immunoperoxidase method, using antiserum to human calcitonin. C-cell nuclei were counted in every sixth section of both left and right lobes. The average number of C-cells counted in the thyroid glands of 8-week-old animals was 18-fold, 5.5-fold and 2.5-fold greater than the number observed in 1-day, 2-week and 4-week-old animals, respectively. C-cell concentration was found to be greatest in 4-week-old mice. Mitoses of C-cells were observed in animals which were 1 day, 2 weeks and four weeks old. No mitotic figures were seen in 8-week-old animals. A few C-cells were seen in close association with neurons. The volume of the thyroid glands of 8-week-old animals was about 14-, 4- and 3-fold greater than the volume in the 1-day-old, 2-week-old and 4-week-old mice, respectively. These changes in the C-cell population during development provide a model for the study of C-cell proliferation and storage of calcitonin.

Human thyroid glands obtained within 2.5 hours of death were examined for the presence and distribution of calcitonin-containing cells using horseradish peroxidase as an indicator in an indirect immunohistochemical procedure. The glands were cut into 10 to 20 transverse slices per lobe and fixed in glutaraldehyde. A representative section of each paraffin-embedded slice was processed and systematically scanned for calcitonin-containing cells. Of 13 glands examined, ten contained calcitonin cells. The cells were found mostly in the follicular epithelium both singly and in groups. They were most numerous in the central region of each lobe of the gland. The isthmus and poles were devoid of calcitonin cells and only occasionally were these cells found at the surface. Parathyroid glands were examined by the same procedure for the presence of calcitonin cells but none were observed. These results demonstrate that calcitonin-containing cells are found regularly in human thyroid glands and that the distribution of these cells is centered in the central region of each lobe of the gland.

Summary Four cases in which the ultimobranchial body (UB) failed to fuse with the median thyroid primordium were observed. In these cases calcitonin-containing cells were only found in the discrete UB which was found at the dorso-lateral edge of the cricoid cartilage and in two cases had a diverticulum directed toward the cricothyroid ligament.

A method for the histochemical demonstration of lactic dehydrogenase (LDH) is presented. This method utilizes a semipermeable membrane to minimize enzyme diffusion and high tetrazole concentration with a controlled reaction rate to minimize product diffusion. It was demonstrated that phenazine methosulfate could be used to make the reaction independent of endogenous diaphorase providing either a cytochrome oxidase inhibitor or strict anaerobiosis is employed. Differentiation of cells containing predominantly H-LDH from cells containing predominantly M-LDH was achieved by the use of 4 molal urea or pyruvate in the incubation medium. A preliminary report of the staining patterns observed in rat gastrocnemius and kidney indicates the usefulness of the technique. A new avenue to the study of the distribution of the LDH subunits is thus opened.

The changes in the fractional volume of six structural components in the spleens of Balb/C mice injected with Herpes simplex virus Type 2-transformed cells (H238 tumor cells) were quantitated during progressive tumor growth. Spleen stereology was performed at three time intervals during the early stages of tumor development. The results revealed that the volume of the compact myeloid tissue and reaction center of lymphoid nodules increased about four- to five-fold from 10 to 33 days after H238 tumor cell injection. A progressive increase was also seen in the red pulp volume. Although an increase in volume of the marginal zones around the lymphoid nodules was evident early during the test period, by day 33 the mean value was similar to the control value. These results indicate that the spleen undergoes significant morphological changes in three splenic components during progressive growth of a tumor produced by subcutaneous injection of a virally-transformed cell line.

A refined method for the immunohistological demonstration of neuron-specific enolase (NSE) on 1- to 2-micron Epon-812 section gave characteristic staining of cerebral and cerebellar neurons. This method has made it possible to obtain a more detailed characterization of the heterogeneity of rat pinealocytes in the superficial portion of the rat pineal complex. Thirty adult male rats have been studied, five of which were used in a photometric analysis of the distribution of NSE. Pinealocytes stained either intensely or weakly for the NSE antigen and exhibited an uneven distribution within a given region. Further analysis of the gland revealed a distal to proximal decrease in stain intensity. It is suggested that the more strongly stained cells, being concentrated distally, are under sympathetic control.

The relationships of tibial endosteal osteoclasts to bone surfaces were quantitatively evaluated during initiation of calcium repletion in calcium-deficient rats. To do this, indices of osteoclast-bone relationships obtained by light microscopy were devised and evaluated by comparing with those obtained by electron microscopy (EM). These indices are the percent of the osteoclast width that (1) exhibits markers indicative of a ruffled border, (2) is in close contact with bone, (3) is isolated from bone by other cell types, and (4) is separated from bone by intercellular material. The indices obtained by light microscopy were strongly correlated with similar indices obtained by EM and were equally sensitive but considerably easier to obtain. The ruffled border and contact index were significantly decreased by 3 hours after beginning the meal whereas cells of other types became interposed between the osteoclasts and the bone.

In a study of the effect of pinealectomy on thyroid C-cell number, 8 animals out of 66 were found to have thymic tissue in close association with the thyroid. Cells containing immunoreactive calcitonin were found in all of the thyroids but in only one of the 8 pieces of thymus. These cells found in a piece of thymic tissue associated with the right thyroid lobe were located immediately under the capsule and did not form or associate with follicles. Unlike the other animals the rat with thymic calcitonin cells had an unequal distribution of C-cells between the left and right thyroid lobes, but the total number of thyroidal C-cells was the same as that of the other rats. Since the thymus proper was not examined in these 66 animals, ten additional rats were taken for such a study. Thyroid-associated thymic tissue was found in three of these, but none of these thymi showed any immunoreactive cells.

The present study deals with the functionally enigmatic "synaptic" ribbons and spherules of guinea pig pinealocytes. Whereas the ribbons have been shown to exhibit a 24-hr rhythmicity with low numbers during the day and high numbers at night, very little of a definitive nature is known about the spherules. Sixteen male guinea pigs of the Hartley strain were perfusion fixed, 8 between 0900-1100 hr, and 8 between 2100-2300 hr. The ribbons and spherules were counted in the pineal parenchyma of the proximal, intermediate, and distal regions. In confirmation of earlier studies, it was found that "synaptic" ribbons are equally abundant in the proximal, intermediate, and distal regions of the gland, during both the day and the night, and that they increase significantly in number at night when compared with daytime values. The spherules, by contrast, are more abundant proximally and are present in greater numbers during the day than at night. As ribbons and spherules are usually not found in one and the same pinealocytic profile, and based on previous electrophysiological studies, it is proposed that the ribbons are morphological markers of nocturnally active pinealocytes, whereas spherules characterize diurnally active pinealocytes.

The tissue distribution of enzymatic activities in intestinal metaplasia stomachs exhibiting chronic gastritis was compared histochemically with that of the small intestine in man.

Background Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells. Methods A range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy. Results Significant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions. Conclusion These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.