Immunofluorescence Staining Of Tissue Sections Research Articles

Magnesium Ion-Doped Mesoporous Bioactive Glasses Loaded with Gallic Acid Against Myocardial Ischemia/Reperfusion Injury by Affecting the Biological Functions of Multiple Cells.

Excessive generation of reactive oxygen species (ROS) following myocardial ischemia-reperfusion (I/R) can result in additional death of myocardial cells. The rapid clearance of ROS after reperfusion injury and intervention during subsequent cardiac repair stages are crucial for the ultimate recovery of cardiac function. Magnesium-doped mesoporous bioactive glasses were prepared and loaded with the antioxidant drug gallic acid into MgNPs by sol-gel method. The antioxidant effects of MgNPs/GA were tested for their pro-angiogenic and anti-inflammatory effects based on the release characteristics of GA and Mg2+ from MgNPs/GA. Later, we confirmed in our in vivo tests through immunofluorescence staining of tissue sections at various time points that MgNPs/GA exhibited initial antioxidant effects and had both pro-angiogenic and anti-inflammatory effects during the cardiac repair phase. Finally, we evaluated the cardiac function in mice treated with MgNPs/GA. We provide evidence that GA released by MgNPs/GA can effectively eliminate ROS in the early stage, decreasing myocardial cell apoptosis. During the subsequent cardiac repair phase, the gradual release of Mg2+ from MgNPs/GA stimulated angiogenesis and promoted M2 macrophage polarization, thereby reducing the release of inflammatory factors. MgNPs/GA acting on multiple cell types is an integrated solution for comprehensive attenuation of myocardial ischaemia-reperfusion injury and cardiac function protection.

Platelet membrane-derived biomimetic microbubbles with enhanced targeting ability for the early detection of myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury (MIRI) is a widely recognized cardiovascular disease that significantly impacts the prognosis of patients undergoing myocardial infarction recanalization. This condition can be fatal and involves complex pathophysiological mechanisms. Early diagnosis of MIRI is crucial to minimize myocardial damage and reducing mortality. Based on the inherent relationship between platelets and MIRI, we developed biomimetic microbubbles coated with platelet membrane (MB-pla) for early identification of MIRI. The MB-pla were prepared through a recombination process involving platelet membrane obtained from rat whole blood and phospholipids, blended in appropriate proportions. By coating the microbubbles with platelet membrane, MB-pla acquired various adhesion molecules, thereby gaining the capability to selectively adhere to damaged endothelial cells in the context of MIRI. In vitro experiments demonstrated that MB-pla exhibited remarkable targeting characteristics, particularly toward type IV collagen and human umbilical vein endothelial cells that had been injured through hypoxia/reoxygenation procedures. In a rat model of MIRI, the signal intensity produced by MB-pla was notably higher than that of control microbubbles. These findings were consistent with results obtained from fluorescence imaging of isolated hearts and immunofluorescence staining of tissue sections. In conclusion, MB-pla has great potential as a non-invasive early detection method for MIRI. Furthermore, this approach can potentially find application in other conditions involving endothelial injury in the future.

Carbonic Anhydrase IX Targeting Mn(II)-Based Magnetic Resonance Molecular Imaging Probe for Hypoxia Tumors.

Physiological hypoxic conditions in the tumor microenvironment and consequential overexpression of carbonic anhydrase IX (CA IX) are two characteristics shared by numerous types of solid malignant tumors. Early detection with hypoxia assessment is crucial to improve the prognosis and therapy outcomes of hypoxia tumors. Herein, using acetazolamide (AZA) as a CA IX-targeting moiety, we design and synthesize an Mn(II)-based MR imaging probe (named AZA-TA-Mn) incorporating AZA and two Mn(II) chelates of Mn-TyEDTA on a rigid triazine (TA) scaffold. The per Mn relaxivity of AZA-TA-Mn is 2-fold higher than its monomeric Mn-TyEDTA, which allows it for low-dose imaging of hypoxic tumors. In a xenograft mice model of esophageal squamous cell carcinoma (ESCC), a low dosage of AZA-TA-Mn (0.05 mmol/kg) can selectively produce prolonged and stronger contrast enhancement in the tumor compared to the non-specific Gd-DTPA (0.1 mmol/kg). A competition study of co-injection of free AZA and Mn(II) probes confirms the in vivo tumor selectivity of AZA-TA-Mn, resulting in a more than 2.5-fold decreased tumor-to-muscle contrast-to-noise ratio (ΔCNR) at 60 min post-injection. MR imaging results were further supported by the quantitative analysis of Mn tissue levels, as the co-injection of free AZA resulted in significantly reduced Mn accumulation in tumor tissues. Finally, immunofluorescence staining of tissue sections confirms the positive correlation between the tumor accumulation of AZA-TA-Mn and CA IX overexpression. Hence, using CA IX as the hypoxia biomarker, our results illustrate a practical strategy for the development of novel imaging probes for hypoxic tumors.

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

Intranasal Adeno-Associated Virus Mediated Gene Delivery and Expression of Human Iduronidase in the Central Nervous System: A Noninvasive and Effective Approach for Prevention of Neurologic Disease in Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.

Expansion of Regulatory T Cells Promotes the Clearance of Apoptotic Cells by Macrophages in Murine Atherosclerotic Plaques

A key process in inflammation resolution is efferocytosis, the clearance of apoptotic cells (AC) by phagocytes. Defective efferocytosis has been implicated in diseases characterized by chronic, nonresolving inflammation, including atherosclerosis. In atherosclerotic plaques, the accumulation of uncleared apoptotic cells leads to necrotic core formation, thereby increasing plaque instability, likelihood of rupture, and risk for acute cardiovascular events such as heart attack and stroke. Expansion of regulatory T cells (Tregs) in murine atherosclerosis decreases aortic root lesion area through mechanisms that are not fully understood. Given that Treg coordination of macrophage function has been reported, we hypothesized that expansion of the Treg compartment during advanced atherosclerosis promotes lesional efferocytosis. First, we used zymosan induced peritonitis as a proof‐of‐concept model to interrogate how Tregs may affect the efferocytic capacity of macrophages. Using mice in which the human diphtheria toxin (DT) receptor sequence is knocked into the 3′ UTR of the Treg lineage‐specific gene, Foxp3, we used DT to deplete Tregs in the days following injection with 0.1 mg Zymosan. Twelve days after injury, mice were ip injected with fluorescently labelled apoptotic neutrophils. After 45 minutes, the peritoneal cavity was lavaged and efferocytosis was measured by flow cytometry. Interestingly, uptake of labelled AC by macrophages in Treg‐depleted mice was significantly reduced compared to PBS‐injected control mice (30% vs 52%, p<0.05). Similarly, when assayed ex vivo, efferocytosis was again found to be defective in macrophages harvested from Treg‐depleted mice (p<0.05). Next, to determine whether Tregs could promote efferocytosis in atherosclerotic lesions, low density lipoprotein receptor deficient (Ldlr−/−) mice were placed on a western diet for a total of 15 weeks. Starting at week 12, mice were treated with with an IL‐2/anti‐IL2 antibody complex (IL2C) that is known to specifically expand Foxp3+ regulatory T cells. After three weeks of treatment, Tregs were significantly increased in the spleen and blood of treated, but not control, mice. We investigated efferocytosis in aortic root lesions by immunofluorescence staining of tissue sections to identify the association of macrophages (F4/80+) and apoptotic cells (TUNEL+). Quantification of the ratio of macrophage‐associated to “free” AC revealed that efferocytosis was significantly increased in aortic root lesions of IL2C treated mice (p<0.05). Interleukin‐10 has been reported to be elevated during Treg expansion in athero‐prone mice and is known to be a Treg effector molecule in other settings. To identify whether IL‐10 is an important player in the promotion of efferocytosis during Treg expansion, we treated mice with an IL‐10 neutralizing antibody or IgG during the expansion period. Strikingly, IL‐10 neutralization abrogated the improvement in lesional efferocytosis without affecting Treg expansion. Cumulatively, these data demonstrate a positive relationship between Foxp3+ Tregs and efferocytosis. Future investigations aim to elucidate the signaling mechanism through which this occurs.Support or Funding InformationThis work was supported in part by a T32 training grant from the NIH (HL007343)

706. Non-Invasive Intranasal Administration of AAV9-Iduronidase Prevents Emergence of Neurologic Disease and Neurocognitive Dysfunction in a Murine Model of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAGs). In the severe form of the disease (Hurler syndrome), death results by age 10. Current treatments for this disease include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality.We have taken a novel approach to treat neurologic disease associated with Hurler syndrome, using intranasal administration of an IDUA-encoding AAV9 vector. A CAGS regulated AAV9-IDUA vector was infused intranasally into adult mice (2-3 months of age) that had been immunotolerized at birth with Aldurazyme to prevent anti-IDUA immune response. Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain. QPCR analysis of vector genomes indicated only background levels in all portions of the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain. This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze. Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape. Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals. There was no significant difference between heterozygote animals and treated animals, while latency to escape was significantly different between these two groups and MPS I deficient animals (P<0.001). This novel, non-invasive strategy for intranasal AAVIDUA administration could potentially be used to treat CNS manifestations of several disorders, including lysosomal storage diseases.

Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes

The contractile properties of cardiac myocytes depend on the calcium (Ca2+) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca2+-induced Ca2+ release (100nm) and were grouped into ‘superclusters’. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca2+ levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/μm3) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca2+ signalling.

NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions

The spectrum of cutaneous adverse drug reactions (cADRs) ranges from benign presentations to severe life-threatening forms such as toxic epidermal necrolysis (TEN). In TEN, granulysin has been shown to be the key cytotoxic molecule. Still, little is known about the expression of granulysin in other cADRs. As an important source of granulysin, natural killer (NK) cells are of major interest in cADRs. Recently, NKp46 has been identified as the most selective NK-cell marker. However, the role of NKp46(+) cells in cADRs and their contribution to granulysin expression remain to be elucidated. Immunohistochemical and immunofluorescence staining of tissue sections from multiple cADRs were quantitatively and qualitatively evaluated. Further, in vivo and in vitro drug-stimulation tests were performed. Granulysin is expressed at different levels in multiple cADRs both by NKp46(+) cells and by CD8(+) T cells. Even in mild forms of cADRs, granulysin can be induced in vivo and in vitro in a drug-specific manner. NKp46(+) cells were found to infiltrate the dermal/epidermal junction particularly in TEN. The impressive clinical differences of cADRs may not be uniquely explained by the expression of granulysin. Additional factors such as drug-specific activation and recruitment of NKp46(+) cells to the epidermis may play a role in determining the severity of cADRs. Therefore, unraveling the effects of drugs on NK-cell activation and trafficking may help to better understand the cytotoxic mechanisms behind cADRs.

Breast cancer instructs dendritic cells to express OX40 ligand and to prime pro-inflammatory type 2 CD4+ T cells that facilitate tumor development.

Abstract Abstract #1050 We found that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. CD4+ T cells infiltrating breast cancer tumors secrete type 1 (IFN-g), pro-inflammatory (TNF) as well as high levels of type 2 (IL-4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated STAT6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells.&amp;#x2028; To determine the link between breast cancer, DCs and CD4+ T cells, we implanted human breast cancer cell lines in NOD/SCID b2m-/- mice engrafted with human CD34+ hematopoietic progenitor cells (HPCs) and autologous T cells. There, CD4+ T cells promote early tumor development. This can be prevented by administration of IL-13 antagonists and is dependent on DCs. Furthermore, these humanized mice demonstrate a signature of IL-13.&amp;#x2028; Similarly to patient samples, in humanized mice breast cancer but not melanoma tumors are infiltrated with large numbers of human myeloid DCs. DC isolated from breast tumor and its draining lymph nodes induce the production of high level of IL4, IL13 and TNF by naive CD4 T cells further demonstrating that breast tumor modulates DC to induce an inflammatory Th2 response.&amp;#x2028; Accordingly, immunofluorescence staining of breast cancer tissue sections from patients shows high expression of OX40 ligand on DCs. This can be ascribed to activity of breast cancer cell-derived soluble factors. Finally, blocking OX40 ligand prevents generation of IL-13 secreting CD4+ T cells.&amp;#x2028; Thus, breast cancer targets DCs to generate pro-inflammatory type 2 immunity that promotes its development.&amp;#x2028; Supported by U19 AIO57234, RO-1 CA89440, CA78846, and CA85540. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1050.

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development.

Evidence of expression of endotoxin receptors CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells.

CD14, a GPI-linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern-recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll-like receptor molecules, TLR2 and TLR4, and the associated molecule MD-2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40-transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time- and dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR)-Southern was positive for CD14, TLR2, TLR4 and MD-2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT-PCR) and HP62 cells showed that LPS regulates glucose-dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha]. The functional expression of CD14 and associated molecules in islet beta cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

Expression of Ly-6D on the surface of normal and neoplastic mammary epithelial cells of the mouse.

Rat were immunized with mouse mammary epithelial cells and monoclonal antibodies (MAbs) were obtained to identify antigens stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Examination of the reactivities of the MAbs by immunofluorescence staining of tissue sections showed that one of the antibodies, MAb 2A2, reacted with luminal epithelium of the mammary gland and spontaneous mammary carcinomas of SHN mice. Further examination of the tissue lysates by western blot analysis revealed that MAb 2A2 reacts with a 17‐kDa antigen expressed in normal mammary epithelial cells and mammary carcinoma cells. The antigen recognized by MAb 2A2 was absent in the lysates of liver, lung, salivary gland, kidney, small intestine, ovary and uterus. After immunoaffinity purification of the MAb 2A2‐ recognized antigen and determination of its N‐terminal amino acid sequence, we identified the antigen as Ly‐6D, also known as ThB, which belongs to a family of glycosyl‐phosphatidylinositol‐anchored cell surface proteins. Northern blot analysis further demonstrated that Ly‐6D mRNA is expressed in the mammary gland. Based on these observations we concluded that Ly‐6D is stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Ly‐6D will serve as a useful epithelial cell surface marker for the study of mammary gland development, as well as for breast cancer research.

Expression of Carbonic Anhydrase V in Pancreatic Beta Cells Suggests Role for Mitochondrial Carbonic Anhydrase in Insulin Secretion

Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets.

Basement Membrane Proteoglycans Are of Epithelial Origin in Rodent Skin

Basement membrane proteoglycans in mammalian skin comprise at least one chondroitin sulfate proteoglycan and heparan sulfate proteoglycans, including perlecan. In this study, the origins of basement membrane chondroitin sulfate proteoglycan and perlecan were investigated both in vivo and in vitro. For in vivo experiments, pieces of newborn rat epidermis obtained by dispase treatment were grafted onto athymic nude mice. Three and six weeks after grafting, immunofluorescence analysis of the grafted skin was carried out, using monoclonal antibodies specific for rat basement membrane chondroitin sulfate proteoglycan and rat and mouse perlecan. While the isolated rat epidermis was shown to completely lack rat basement membrane chondroitin sulfate proteoglycan and rat basement membrane heparan sulfate proteoglycans, including perlecan, immunofluorescence staining of tissue sections from the grafted sites on mice demonstrated the presence of rat basement membrane chondroitin sulfate proteoglycan and rat perlecan on interfollicular and follicular basement membranes including that separating dermal papillae from adjacent hair follicle epithelium. In contrast, the basement membranes of all dermal capillaries were positive for mouse perlecan, but negative for rat basement membrane chondroitin sulfate proteoglycan and rat perlecan, including the basement membranes of papillary dermal capillaries beneath the rat epidermis. These data suggest that basement membrane proteoglycans of the dermal-epidermal junction and hair follicle epithelium are of epidermal (epithelial) origin in vivo. Stratified rat keratinocytes cultured on a collagen matrix at the air-liquid interface showed the synthesis of perlecan, laminin 1, and type IV collagen in basement membranes, but not clearly detectable basement membrane chondroitin sulfate proteoglycan.

Vitronectin in Colorectal Adenocarcinoma—Synthesis by Stromal Cells in Culture

We have investigated the expression and cellular source of vitronectin in colorectal adenocarcinoma. Immunofluorescence staining of tissue sections revealed the presence of vitronectin in the stroma of the 11 tumors studied, but not in adjacent normal colon. A method was devised for the isolation from colorectal adenocarcinomas of fibroblast-like cells that stained positive for vimentin but negative for cytokeratin. These tumor-derived stromal cells synthesized and secreted vitronectin, as revealed by metabolic labeling and immunoprecipitation. This was confirmed by Southern blot analysis of polymerase chain reaction amplification products from reverse-transcribed RNA. Normal skin fibroblasts did not synthesize vitronectin. Immunofluorescence staining showed vitronectin deposited at focal contact sites in the tumor-derived cells, where it colocalized with vinculin and the αv integrin subunit. The deposition of vitronectin into focal contact sites was not dependent on the presence of serum. The finding that vitronectin can be synthesized and secreted by tumor-derived fibroblast-like cells in culture indicates that vitronectin expression can be promoted by as yet unknown signals provided in disease states, such as cancer.

Evidence that intestinal IgA plasma cells in μ,ϰ transgenic mice are derived from B-1 (Ly-1 B) cells

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived, Igh-Ca allotype) mu heavy chain and kappa light chain transgenes, specific for trinitrophenyl, on a C57BL background (Igh-Cb allotype). FACS analyses show that the majority of B cells in peripheral lymphoid organs and bone marrow (BM) express transgenic IgM exclusively. A small proportion of the B cells, however, express endogenous IgM, usually concomitant with transgenic IgM. Three criteria establish that the endogenous IgM expressing B cells belong to the B-1 cell lineage. (i) Endogenous IgM expressing B cells in B6-Sp6 mice have the same localization pattern as B-1 cells from normal animals: they are enriched in the peritoneal cavity. (ii) The endogenous IgM+ B cells have the phenotype of B-1 cells: the endogenous IgM+ peritoneal B cells express Mac-1 (CD11b) and low levels of IgD, and most also express CD5 (Ly-1). (iii) B6-Sp6 BM poorly reconstitutes endogenous IgM+ B cells, just as adult BM from normal mice poorly reconstitutes B-1 cells. In contrast, B cells which only express the transgene are readily reconstituted by B6-Sp6 BM. The few endogenous IgM+ cells in the B6-Sp6 BM recipients are located in the peritoneal cavity and have the phenotype of B-1b cells (previously the Ly-1 B sister population), which are known to be reconstituted by adult BM. Two-color immunofluorescence staining of tissue sections from the gut and from isolated gut lamina propria cells shows the presence of many IgA containing cells, about one-third of which simultaneously express cytoplasmic (transgenic) IgM. The C-region of this IgA is produced by endogenous C alpha genes, because the transgene encodes only for C mu. Furthermore, the majority of gut IgA containing cells do not express the idiotype of the transgene, indicating that most of the gut IgA cells are encoded by endogenous VH genes and thus the result of an isotype switch from endogenous IgM expressing B cells. Since the endogenous IgM+ cells are B-1 cells (both B-1a and B-1b), the data strongly indicate that the intestinal IgA plasma cells also belong to the B-1 cell lineage.

Renewal of enterochromaffin cells in the rat caecum.

The localization, morphology, and neurohormonal peptide content of neuroendocrine cells have been extensively investigated. Relatively little is known about the kinetics of growth and differentiation of these cells. We studied the kinetics of enterochromaffin (EC) cells in the caecum of the rat, by applying the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU), to identify cells in S-phase, administered in pulse-chase and synchronous continuous labeling experiments. By double indirect immunofluorescence staining of tissue sections, using antibodies against serotonin and BrdU, percentages of BrdU positive EC cells could be enumerated, from which cell-kinetic parameters were derived. The following conclusions were drawn: 1) EC cells are renewed by proliferation of EC cells and by recruitment from proliferating precursor cells. 2) Caecal EC cells appear to consist of a relatively rapidly renewing and migrating fraction (60-65%) with a turnover time of approximately 16 days and a relatively slowly renewing and possibly stationary fraction (35-40%) with an estimated turnover time of approximately 150 days. 3) Seventy percent of the EC cells are localized in the lower half of mucosal crypts, 30% in the upper half. After prolonged labeling the percentage of labeled EC cells in the lower crypt half always exceeds that in the upper crypt half. This decrease in labeled EC cells during migration towards the mucosal surface indicates loss of endocrine cells, possibly owing to loss of endocrine characteristics.

Development of resistance to coccidiosis in the absence of merogonic development using X-lrradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that Sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.

Differentiation-Dependent Expression of Keratins in Human Oral Epithelia

The polypeptide composition of epithelial keratins varies with the state of differentiation. The epithelia lining the human oral cavity show regional variations in their histology. In the present study, paired samples of nonkeratinized buccal epithelium and keratinized hard palate epithelium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblots with monoclonal antibodies AE1, AE2, and AE3, and results were correlated with immunofluorescence staining of tissue sections of the same samples. Keratins from hard palate (Mr 67K, 63-65K, 58K, 56.5K, 56K, 50K, 48K) and epidermis (Mr 67K, 63-65K, 58K, 56.5K, 50K) were similar to each other but distinctly different from those of buccal epithelium (major bands of Mr 52K and 59K, minor bands of 50K and 58K). The immunoblot analysis further indicated the similarity of hard palate and epidermal keratins, in contrast to those of buccal epithelium. Each oral tissue expressed keratins of the type I (AE1, acidic) subfamily and type II (AE3, basic) subfamily. In tissue sections, the predominant staining pattern for nonkeratinized buccal epithelium was: AE1, positive in the basal layer; AE2, negative; AE3, positive in all layers. In contrast, the staining pattern for keratinized palatal epithelium was: AE1 and AE2, positive in the suprabasal layers; AE3, positive in all layers. Strong suprabasal AE1 staining in palate may be related to the presence of the 48K keratin. Some buccal samples showed an alternate staining pattern of spotty suprabasal staining with AE1 and AE2 which was correlated with the expression of the 56.5K and 63-67K keratins, as well as filaggrin. These results suggest differentiation-specific expression of the keratins and show immunologically detectable variation in the apparently normal differentiation pattern of nonkeratinized buccal epithelium.