Jan Marc Orenstein

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral–genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 °C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.

Complex extracellular matrices promote tissue-specific stem cell differentiation.

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA‐developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin‐1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular‐ and tubular‐like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular‐ and glandular‐like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue–stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.

Characterization of Encephalitozoon (Septata) intestinalis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients

ABSTRACT. Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK‐13, MDCK, HT‐29, Caco‐2, Vero, and 1047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane‐bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction‐based methods with restriction endonuclease digestion, double‐stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.

Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research

In transmission electron microscopy (TEM), electrons are transmitted through a plastic-embedded specimen, and an image is formed. TEM enables the resolution and visualization of detail not apparent via light microscopy, even when combined with immunohistochemical analysis. Ultrastructural examination of tissues, cells and microorganisms plays a vital role in diagnostic pathology and biologic research. TEM is used to study the morphology of cells and their organelles, and in the identification and characterization of viruses, bacteria, protozoa and fungi. In this protocol, we present a TEM method for preparing specimens obtained in clinical or research settings, discussing the particular requirements for tissue and cell preparation and analysis, the need for rapid fixation and the possibility of analysis of tissue already fixed in formalin or processed into paraffin blocks. Details of fixation, embedding and how to prepare thin and semi-thin sections, which can be used for analysis complementary to that performed ultimately using TEM, are also described.

TGF-β influences the life and death decisions of T lymphocytes

Abstract TGF-β is a powerful mediator of immune cell phenotype and function. In TGF-β1 homozygous null mice, aberrant regulation of the immune response culminates in lethal cardiopulmonary inflammation. In dissecting the underlying mechanisms leading to the attack of self, a role for TGF-β1 in controlling apoptosis and T cell selection patterns was uncovered. Increased levels of apoptosis and TCR mediated cell death disrupted normal negative and positive T cell selection in the thymus. Moreover, in peripheral T cell populations, increased T lymphocyte death was associated with increased expression of apoptosis-inducing receptors. Persistent activation of T cells engendered unchecked apoptosis which, rather than reducing, further exacerbated, tissue inflammation due to the absence of TGF-β1. TGF-β, normally generated by macrophages during clearance of apoptotic cells contributes to dampening of inflammatory sequelae associated with phagocytosis. Collectively, these data demonstrate a pivotal role for TGF-β in multiple stages of T cell apoptosis, selection, activation and clearance.

The Macrophage in HIV Infection

Macrophages play a key role in several critical aspects of HIV disease. They appear to be the first cells infected by HIV and perhaps the very source of HIV production when CD4+ cells are markedly depleted in the patient. Macrophages and microglial cells are the cells infected by HIV in the CNS. In tonsils and adenoids of HIV-infected patients, macrophages fuse into multinucleated giant cells that produce copious amounts of virus. Finally, opportunistic pathogens can cause an upregulation of HIV production by macrophages, often in the multinucleated form.

Opportunistic disorders of the gastrointestinal tract in the age of highly active antiretroviral therapy.

Since the 1996 advent of highly active antiretroviral therapy (HAART) for the treatment of HIV/AIDS, the incidence of most opportunistic disorders in the developed world has dramatically declined but definitely has not disappeared. The number of new yearly HIV infections (about 55,000) and the total number of US infections (more than 1.1 million) remain very significant. Post-HAART gastrointestinal (GI) symptoms and biopsy results are still common, especially in large inner-city hospitals. The same opportunistic GI disorders were diagnosed in 442 endoscopies performed since 1996 as before, but at about one half the rate. The esophagus had the highest rate of positive biopsy results (46%), especially due to Candida. Helicobacter pylori infection has become the most common gastric infection. The small bowel still showed cytomegalovirus (CMV), cryptosporidia, and Mycobacterium avium complex (MAC) infections. In decreasing order, the most common large bowel infections were CMV, cryptosporidiosis, MAC, and spirochetosis. Cases of adenovirus, bacterial colitis, Kaposi sarcoma, and lymphoma were still diagnosed. Rectal biopsy specimens were the least productive. Microsporidiosis is now being diagnosed with special stains. Thus, where HIV/AIDS is common, it is important to be able to diagnose these GI processes. In addition to presenting post-HAART incidences, diagnostic features and aids are described for selected entities.

Fatal Pulmonary Microsporidiosis Due to Encephalitozoon cuniculi Following Allogeneic Bone Marrow Transplantation for Acute Myelogenous Leukemia

Microsporidia are ubiquitous obligate eukaryotic intracellular parasites that are now felt to be more akin to degenerate fungi than to protozoa. Microsporidia can be highly pathogenic, causing a broad range of symptoms in humans, especially individuals who are immunocompromised. The vast majority of human cases of microsporidiosis have been reported during the past 20 years, in patients with HIV/AIDS, while only relatively rare cases have been described in immunocompetent individuals. However, microsporidia infections are being increasingly reported in patients following solid-organ transplanation, where the main symptom has been diarrhea. The authors report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the United States and only the second case in the world. The patient, with a history of Hodgkin disease followed by acute myelogenous leukemia received a T-cell-depleted graft, but succumbed to respiratory failure 63 days post transplantation. An open lung biopsy, taken just before death, was originally thought to show toxoplasmosis. The correct diagnosis of microsporidiosis was made postmortem by light and electron microscopy. DNA polymerase chain reaction analysis confirmed the diagnosis and furthermore revealed it to be the dog strain of the microsporidia species Encephalitozoon cuniculi. Although to date rarely diagnosed, microsporidial infection should also be considered in the differential diagnosis of, e.g., unexplained pulmonary infection in bone marrow transplant patients.

The tissue microarray data exchange specification: implementation by the Cooperative Prostate Cancer Tissue Resource

BackgroundTissue Microarrays (TMAs) have emerged as a powerful tool for examining the distribution of marker molecules in hundreds of different tissues displayed on a single slide. TMAs have been used successfully to validate candidate molecules discovered in gene array experiments. Like gene expression studies, TMA experiments are data intensive, requiring substantial information to interpret, replicate or validate. Recently, an open access Tissue Microarray Data Exchange Specification has been released that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. While this specification provides sufficient information for the reproduction of the experiment by outside research groups, its initial description did not contain instructions or examples of actual implementations, and no implementation studies have been published. The purpose of this paper is to demonstrate how the TMA Data Exchange Specification is implemented in a prostate cancer TMA.ResultsThe Cooperative Prostate Cancer Tissue Resource (CPCTR) is funded by the National Cancer Institute to provide researchers with samples of prostate cancer annotated with demographic and clinical data. The CPCTR now offers prostate cancer TMAs and has implemented a TMA database conforming to the new open access Tissue Microarray Data Exchange Specification. The bulk of the TMA database consists of clinical and demographic data elements for 299 patient samples. These data elements were extracted from an Excel database using a transformative Perl script. The Perl script and the TMA database are open access documents distributed with this manuscript.ConclusionsTMA databases conforming to the Tissue Microarray Data Exchange Specification can be merged with other TMA files, expanded through the addition of data elements, or linked to data contained in external biological databases. This article describes an open access implementation of the TMA Data Exchange Specification and provides detailed guidance to researchers who wish to use the Specification.

Pulmonary infection with microsporidia after allogeneic bone marrow transplantation

Summary:Microsporidia are obligate, intracellular protozoal parasites that can be pathogenic in immunocompromised individuals. The majority of cases of microsporidiosis have been documented in patients with HIV, and only a few case reports exist of infection in solid organ transplant patients. We report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the US. The patient was a recipient of a T-cell-depleted graft who succumbed to complications from respiratory failure 63 days post transplant. The diagnosis was made post mortem by electron microscopy and confirmed with PCR. Although rare, microsporidial infection should be considered in the differential diagnosis of unexplained pulmonary infection in bone marrow transplant patients.