Tunneled island pedicle flap reconstruction for upper lateral cutaneous lip defects.

BACKGROUND: Esthetic upper lateral cutaneous lip reconstruction preserves the apical triangle, nasolabial fold symmetry, and free margin position. The tunneled island pedicle flap (IPF) is a novel single-stage reconstruction to achieve these goals. OBJECTIVES: Describe the technique and patient and surgeon-reported outcomes for the tunneled IPF reconstruction of upper lateral cutaneous lip defects. METHODS: Retrospective chart review of consecutive tunneled IPF reconstruction following Mohs micrographic surgery (MMS) at a tertiary care center between 2014 and 2020. Patients rated their scars using the validated Patient Scar Assessment Scale (PSAS), and independent surgeons rated scars using the validated Observer Scar Assessment Scale (OSAS). Descriptive statistics were generated for patient demographics and tumor defect characteristics. RESULTS: Twenty upper lateral cutaneous lip defects were repaired with the tunneled IPF. Surgeons rated scars with a composite OSAS score of 11.83 ± 4.29 (mean, SD) [scale of 5 (normal skin) to 50 (worst scar imaginable)] and an overall scar score of 2.81 ± 1.11 [scale of 1 (normal skin) to 10 (worst scar imaginable)]. Patients rated their scars with a composite PSAS score of 10 ± 5.39 [scale of 6 (best possible score) to 60 (worst)] and with an overall score of 2.2 ± 1.78 [scale of 1 (normal skin) and 10 (very different from normal skin)]. One flap was surgically revised for pincushioning, but none experienced necrosis, hematoma, or infection. CONCLUSIONS: The tunneled IPF is a single-stage reconstruction for upper lateral cutaneous lip defects with favorable scar ratings by patients and observers.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.

BACKGROUND: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. METHODS: Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. RESULTS: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). CONCLUSION: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene.

BACKGROUND: There is a need to develop robust and clinically applicable gene expression signatures. Hypoxia is a key factor promoting solid tumour progression and resistance to therapy; a hypoxia signature has the potential to be not only prognostic but also to predict benefit from particular interventions. METHODS: An approach for deriving signatures that combine knowledge of gene function and analysis of in vivo co-expression patterns was used to define a common hypoxia signature from three head and neck and five breast cancer studies. Previously validated hypoxia-regulated genes (seeds) were used to generate hypoxia co-expression cancer networks. RESULTS: A common hypoxia signature, or metagene, was derived by selecting genes that were consistently co-expressed with the hypoxia seeds in multiple cancers. This was highly enriched for hypoxia-regulated pathways, and prognostic in multivariate analyses. Genes with the highest connectivity were also the most prognostic, and a reduced metagene consisting of a small number of top-ranked genes, including VEGFA, SLC2A1 and PGAM1, outperformed both a larger signature and reported signatures in independent data sets of head and neck, breast and lung cancers. CONCLUSION: Combined knowledge of multiple genes' function from in vitro experiments together with meta-analysis of multiple cancers can deliver compact and robust signatures suitable for clinical application.

rHVDM: an R package to predict the activity and targets of a transcription factor.

SUMMARY: Highly parallel genomic platforms like microarrays often present researchers with long lists of differentially expressed genes but contain little or no information on how these genes are regulated. rHVDM is a novel R package which uses gene expression time course data to predict the activity and targets of a transcription factor. In the first step, rHVDM uses a small number of known targets to derive the activity profile of a given transcription factor. Then, in a subsequent step, this activity profile is used to predict other putative targets of that transcription factor. A dynamic and mechanistic model of gene expression is at the heart of the technique. Measurement error is taken into account during the process, which allows an objective assessment of the robustness of fit and, therefore, the quality of the predictions. The package relies on efficient algorithms and vectorization to accomplish potentially time consuming tasks including optimization and differential equation integration. We demonstrate the efficiency and accuracy of rHVDM by examining the activity of the tumour-suppressing transcription factor, p53. AVAILABILITY: The version of the package presented here (1.8.1) is freely available from the Bioconductor Web site (http://bioconductor.org/packages/2.3/bioc/html/rHVDM.html).

Protective attenuated lentivirus immunization induces SIV-specific T cells in the genital tract of rhesus monkeys.

Live attenuated lentivirus immunization is the only vaccine strategy that elicits consistent protection against intravaginal challenge with pathogenic simian immunodeficiency virus (SIV). To determine the mechanism of protection in rhesus monkeys infected with attenuated simian-human immunodeficiency virus (SHIV)89.6, a detailed analysis of SIV Gag-specific T-cell responses in several tissues including the genital tract was performed. Six months after SHIV infection, antiviral T-cell responses were rare in the cervix; however, polyfunctional, cytokine-secreting, and degranulating SIV Gag-specific CD4(+) T cells were consistently found in the vagina of the immunized macaques. SIV-specific CD8(+) T cells were also detected in the vagina, blood, and genital lymph nodes of most of the animals. Thus, an attenuated SHIV vaccine induces persistent antiviral T cells in tissues, including the vagina, where these effector T-cell responses may mediate the consistent protection from vaginal SIV challenge observed in this model.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

TPD52 and NFKB1 gene expression levels correlate with G2 chromosomal radiosensitivity in lymphocytes of women with and at risk of hereditary breast cancer.

PURPOSE: To evaluate a transcriptomic approach to identify healthy women at increased risk of breast cancer due to G2-radiosensitivity and look at transcripts that are differentially expressed between individuals. MATERIALS AND METHODS: We perform the first study to assess the association of G2 radiosensitivity with basal gene expression in cultured T-lymphocytes from 11 women with breast cancer and 12 healthy female relatives using Affymetrix GeneChips. RESULTS: Transcripts associated with radiosensitivity and breast cancer risk were predominantly involved in innate immunity and inflammation, such as interleukins and chemokines. Genes differentially expressed in radiosensitive individuals were more similarly expressed in close family members than in un-related individuals, suggesting heritability of the trait. The expression of tumour protein D52 (TPD52), a gene implicated in cell proliferation, apoptosis, and vesicle trafficking was the most strongly correlated with G2 score while nuclear factor (kappa)-B (NFKB1) was highly inversely correlated with G2 score. NFKB1 is known to be activated by irradiation and its inhibition has been previously shown to increase radiosensitivity. CONCLUSIONS: Gene expression analysis of lymphocytes may provide a quantitative measure of radiation response potential and is a promising marker of breast cancer susceptibility.

Effects of oestrogen on gene expression in epithelium and stroma of normal human breast tissue.

Oestrogen (E) is essential for normal and cancer development in the breast, while anti-oestrogens have been shown to reduce the risk of the disease. However, little is known about the effect of E on gene expression in the normal human breast, particularly when the epithelium and stroma are intact. Previous expression profiles of the response to E have been performed on tumour cell lines, in the absence of stroma. We investigated gene expression in normal human breast tissue transplanted into 9-10-week-old female athymic nude (Balb/c nu/nu) mice. After 2 weeks, when epithelial proliferation is minimal, one-third of the mice were treated with 17beta-oestradiol (E2) to give human luteal-phase levels in the mouse, which we have previously shown to induce maximal epithelial cell proliferation. RNA was isolated from treated and untreated mice, labelled and hybridized to Affymetrix HG-U133A (human) GeneChips. Gene expression levels were generated using BioConductor implementations of the RMA and MAS5 algorithms. E2 treatment was found to represent the largest source of variation in gene expression and cross-species hybridization of mouse RNA from xenograft samples was demonstrated to be negligible. Known E2-responsive genes (such as TFF1 and AREG), and genes thought to be involved in breast cancer metastasis (including mammoglobin, KRT19 and AGR2), were upregulated in response to E treatment. Genes known to be co-expressed with E receptor alpha in breast cancer cell lines and tumours were both upregulated (XBP-1 and GREB1) and downregulated (RARRES1 and GATA3). In addition, genes that are normally expressed in the myoepithelium and extracellular matrix that maintain the tissue microenvironment were also differentially expressed. This suggests that the response to oestrogen in normal breast is highly dependent upon epithelial-stromal/myoepithelial interactions which maintain the tissue microenvironment during epithelial cell proliferation.

Co-immunization of rhesus macaques with plasmid vectors expressing IFN-gamma, GM-CSF, and SIV antigens enhances anti-viral humoral immunity but does not affect viremia after challenge with highly pathogenic virus.

To investigate the adjuvant capacity of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN-gamma), we cloned these rhesus cytokines into a mammalian expression vector. Two groups of six rhesus macaques (Macaca mulatta) received intradermal immunizations of plasmid DNA coding for SIV Eng and Gag, and influenza virus nucleoprotein (Flu-NP), with or without the co-administration of plasmid DNA coding for these cytokines. Humoral immune responses to antigens of both of these viruses and SIV specific T cell proliferative responses were significantly enhanced by co-immunization with the cytokines. These twelve monkeys, and a group of six naive controls, were challenged by the oral mucosal route with the uncloned and highly pathogenic SIVmac251. All monkeys became infected. The early CD4 decline was reduced in the group co-immunized with cytokine and viral plasmids. Unexpectedly, plasma viremia set points were not different in this co-immunized group and the non-immunized control group. On the other hand, monkeys vaccinated with equivalent amounts of empty vector plasmid (i.e. no cytokine inserts) along with plasmids expressing viral antigens demonstrated a slight but significant decrease in acute viremia compared to non-immunized controls (P<0.02). However, viral loads at set points were not significantly different between both the immunized and the non-immunized control group. Thus, although the cytokine vectors demonstrated detectable enhancement of the immune response to different viral antigens, such enhanced response did not translate into better anti-viral control in our experiment. These results underscore the need for further testing of cytokines as vaccine adjuvants in relevant animal models.

The strength of B cell immunity in female rhesus macaques is controlled by CD8+ T cells under the influence of ovarian steroid hormones.

To understand more clearly how mucosal and systemic immunity is regulated by ovarian steroid hormones during the menstrual cycle, we evaluated the frequency of immunoglobulin- and antibody-secreting cells (ISC, AbSC) in genital tract and systemic lymphoid tissues of normal cycling female rhesus macaques. The frequency of ISC and AbSC was significantly higher in tissues collected from animals in the periovulatory period of the menstrual cycle than in tissues collected from animals at other stages of the cycle. The observed changes were not due to changes in the relative frequency of lymphocyte subsets and B cells in tissues, as these did not change during the menstrual cycle. In vitro, progesterone had a dose-dependent inhibitory effect, and oestrogen had a dose-dependent stimulatory effect on the frequency of ISC in peripheral blood mononuclear cell (PBMC) cultures. The in vitro effect of progesterone and oestrogen on ISC frequency could not be produced by incubating enriched B cells alone with hormone, but required the presence of CD8+ T cells. Following oestrogen stimulation, a CD8+ enriched cell population expressed high levels of IFN-gamma and IL-12. The changes in B cell Ig secretory activity that we document in the tissues of female rhesus macaques during the menstrual cycle is due apparently to the action of ovarian steroid hormones on CD8+ T cells. Thus, CD8+ T cells control B cell secretory activity in both mucosal and systemic immune compartments. Understanding, and eventually manipulating, the CD8+ regulatory cell-B cell interactions in females may produce novel therapeutic approaches for autoimmune diseases and new vaccine strategies to prevent sexually transmitted diseases.

Tracking prostate carcinoma micrometastasis to multiple organs using histochemical marker genes and novel cell systems.

Studies of human prostate carcinoma (PCA) have been hampered by only a few cell systems from already-metastatic human disease. We have developed a novel cell system by using tissue cultured CWR22R cells from a xenograft of a primary tumor from a human patient. These cells were transfected with the bacterial lacZ gene to maximize their detection during progression and metastasis in nude mice. LZ-CWR22R cells are extremely stable for lacZ expression over 25 passages and metastasize to lung, liver, and bone from the subcutis - major sites of metastasis of the human disease. A matrigel vehicle facilitated development of primary tumors and micrometastases in all organs. While some micrometastases developed into overt metastases, others remained as micrometastases for long periods of time, possibly providing a model of latency of metastatic disease. An experimental metastasis model (tail vein injection) also generated micrometastases in lung, liver, and bone with differing kinetics of formation and stability. Serial sections of many individual lung micrometastases within one hour of injection indicated considerable heterogeneity in cellular composition (from 1 to 19 cells/site) while liver sites at later times were comprised of only 1 or 2 cells (the size of bone sites were comparable to those of liver). By combining use of these histochemically-tagged PCA cell systems with high resolution molecular analyses (laser-capture microdissection), it will now be possible to analyze gene expression patterns characteristic of micrometastases developing in several different organs.

Evidence for novel functions of the keratin tail emerging from a mutation causing ichthyosis hystrix.

Unraveling the molecular basis of inherited disorders of epithelial fragility has led to understanding of the complex structure and function of keratin intermediate filaments. Keratins are organized as a central alpha-helical rod domain flanked by nonhelical, variable end domains. Pathogenic mutations in 19 different keratin genes have been identified in sequences corresponding to conserved regions at the beginning and end of the rod. These areas have been recognized as zones of overlap between aligned keratin proteins and are thought to be crucial for proper assembly of keratin intermediate filaments. Consequently, all keratin disorders of skin, hair, nail, and mucous membranes caused by mutations in rod domain sequences are characterized by perinuclear clumping of fragmented keratin intermediate filaments, thus compromising mechanical strength and cell integrity. We report here the first mutation in a keratin gene (KRT1) that affects the variable tail domain (V2) and results in a profoundly different abnormality of the cytoskeletal architecture leading to a severe form of epidermal hyperkeratosis known as ichthyosis hystrix Curth-Macklin. Structural analyses disclosed a failure in keratin intermediate filament bundling, retraction of the cytoskeleton from the nucleus, and failed translocation of loricrin to the desmosomal plaques. These data provide the first in vivo evidence for the crucial role of a keratin tail domain in supramolecular keratin intermediate filament organization and barrier formation.

Knowledge, attitudes, and practices related to breast cancer screening: a survey of Arabic women.

BACKGROUND: Incorporating breast cancer screening into day-to-day clinical care leads to early diagnosis and decreases mortality. Patients' participation in screening depends on their knowledge and attitudes, other barriers, and physician behavior. METHODS: A cross-sectional questionnaire survey was conducted to evaluate knowledge, attitudes, barriers, and practices related to breast cancer screening among Arabic women. A convenience sample was selected from 1,750 women aged 40-65 years who, for any reason, attended primary health care (PHC) clinics in Al-Ain, United Arab Emirates (UAE). RESULTS: Of the 1,750 invited women, 1,445 agreed to participate; 78 were excluded from analysis because of histories of breast cancer. Breast self-examination (BSE) was practiced by 12.7% of the study population, clinical breast examination (CBE) by 13.8%, and mammography by 10.3%. Knowledge about breast cancer screening was low in the study population. Women were infrequently instructed about or offered screening for breast cancer by health professionals. Being employed was an independent predictor for participation in the three screening examinations. CONCLUSIONS: Health workers infrequently offered screening examinations and women lacked adequate knowledge about breast cancer screening. Acquired information about barriers to screening may help in the design of effective screening programs for Arabic women.

Anatomic site and immune function correlate with relative cytokine mRNA expression levels in lymphoid tissues of normal rhesus macaques.

Reverse transcriptase real-time polymerase chain reaction was used to determine pro-inflammatory, anti-viral and immunoregulatory cytokine mRNA expression levels in peripheral blood mononuclear cells (PBMC) of healthy juvenile, adolescent and adult rhesus macaques. Few age-related changes in cytokine mRNA expression levels were observed. Expression of interleukin 2 and Mx, a type I interferon-inducible gene, decreased with age, whereas interleukin 4 and macrophage inflammatory protein 1 (MIP-1) alpha and beta mRNA levels increased in older monkeys. Independent of age, the pro-inflammatory cytokines [tumour necrosis factor alpha (TNF-alpha) and chemokines] were expressed at higher mRNA levels in PBMC than the immunoregulatory cytokines (interleukins 2, 4, 12). Pro-inflammatory cytokine mRNA expression levels were highest in lymphoid tissues draining mucosal surfaces. Thus, a correlation exists between cytokine mRNA levels in lymphoid tissues and the anatomical site.

Replication of simian immunodeficiency virus (SIV) in ex vivo lymph nodes as a means to assess susceptibility of macaques in vivo.

Six macaques, apparently uninfected, following low-dose exposure to the pathogenic SIV(mac251) and SIV(SME660) by the mucosal route, were used in a pilot study to investigate whether infectability of ex vivo lymph nodes could predict resistance and/or susceptibility to SIV infection in vivo. Of six macaques exposed to the less-pathogenic virus SIV(MNE), four resisted viral infection. Analysis of the susceptibility of the PBMC of these four animals before SIV(MNE) challenge indicated that all of them were resistant to infection by the SIV(BK28) isolate and, in three of them, this resistance was dependent on CD8+ T cells. Blocks of lymph nodes of these four macaques were resistant to SIV(MNE) infection ex vivo following SIV(MNE) viral challenge exposure. However, the same blocks from the same animals were permissive to the more virulent SIV(251(32H)). Accordingly, three of these macaques were readily infected following challenge exposure with SIV(251(32H)). Lymphoproliferative responses in blood or lymph nodes, local C-C chemokine production in the lymph-node explants, and cytotoxic T-cell activity measured throughout the study did not correlate with ex vivo resistance or susceptibility to in vivo infection. In conclusion, PBMC and lymph-node resistance or susceptibility to infection ex vivo appeared to correlate with in vivo infectivity and, thus, these approaches should be further tested for their predictive value for in vivo infection.

Simian-human immunodeficiency virus containing a human immunodeficiency virus type 1 subtype-E envelope gene: persistent infection, CD4(+) T-cell depletion, and mucosal membrane transmission in macaques.

The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) determines several viral properties (e.g., coreceptor usage, cell tropism, and cytopathicity) and is a major target of antiviral immune responses. Most investigations on env have been conducted on subtype-B viral strains, prevalent in North America and Europe. Our study aimed to analyze env genes of subtype-E viral strains, prevalent in Asia and Africa, with a nonhuman primate model for lentivirus infection and AIDS. To this end, we constructed a simian immunodeficiency virus/HIV-1 subtype-E (SHIV) recombinant clone by replacing the env ectodomain of the SHIV-33 clone with the env ectodomain from the subtype-E strain HIV-1(CAR402), which was isolated from an individual in the Central African Republic. Virus from this recombinant clone, designated SHIV-E-CAR, replicated efficiently in macaque peripheral blood mononuclear cells. Accordingly, juvenile macaques were inoculated with cell-free SHIV-E-CAR by the intravenous or intravaginal route; virus replicated in these animals but did not produce hematological abnormalities. In an attempt to elicit the pathogenic potential of the recombinant clone, we serially passaged this viral clone via transfusion of blood and bone marrow through juvenile macaques to produce SHIV-E-P4 (fourth-passage virus). The serially passaged virus established productive infection and CD4(+) T-cell depletion in juvenile macaques inoculated by either the intravenous or the intravaginal route. Determination of the coreceptor usage of SHIV-E-CAR and serially passaged SHIV-E-P4 indicated that both of these viruses utilized CXCR4 as a coreceptor. In summary, the serially passaged SHIV subtype-E chimeric virus will be important for studies aimed at developing a nonhuman primate model for analyzing the functions of subtype-E env genes in viral transmission and pathogenesis and for vaccine challenge experiments with macaques immunized with HIV-1 env antigens.

Antigen-dependent cytokine mRNA expression by individual rhesus macaque T helper cells by flow cytometry.

Specific patterns of cytokine secretion by CD4(+) T helper (Th) cells determine the nature of immune effector responses. Using a multiparameter, flow cytometric fluorescent in situ hybridization (FISH) assay that detected cytoplasmic mRNA within intact cells, we assessed antigen-specific cytokine expression in rhesus macaque Th cells. In the peripheral lymphocytes of immunized rhesus macaques, FISH detected antigen-induced cytokine gene expression in single Th cells. Analysis of simultaneous cytokine expression by single cells demonstrated that the recall immune response consisted of Th cells expressing either a Th1 (IL-2(+)/IFN-gamma(+)) or a Th2 (IL-4(+)/IL-6(+)) cytokine pattern. In addition to the classic Th subsets, Th cells expressing only one of two Th1 or Th2 defining cytokines were common following antigen restimulation. The data gathered with the FISH assay suggest that, in primates, the immune response to recall antigens consists of nonclassic Th cells, as well as a mixture of polarized Th1 and Th2 T cells.