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PubMed2004

Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors.

The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

Cell Adhesion↗
PubMed2001

Modulation of cell-substrate adhesion by arachidonic acid: lipoxygenase regulates cell spreading and ERK1/2-inducible cyclooxygenase regulates cell migration in NIH-3T3 fibroblasts.

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute approximately 25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

3T3 Cells↗
PubMed1991

Intrahemispheric and interhemispheric spread of cerebral cortical myoclonic activity and its relevance to epilepsy.

Nine patients with cortical myoclonus (due to various pathologies), in whom movement of one limb induced bilateral limb jerks, were investigated. Three of these patients also had bilateral cortical reflex myoclonus when one limb was subjected to an electrical stimulus. The relative latencies to onset of electromyogram (EMG) activity in various ipsilateral and contralateral muscles in action and reflex jerks were studied. Bilateral reflex and action jerks induced by unilateral electrical stimuli or limb movement were not synchronous. EMG activity was usually recorded in the muscles of the stimulated or moved limb, before being recorded in the homologous muscles of the contralateral limb. It is proposed that this difference in relative latency between homologous muscles represents the interhemispheric delay due to the transcallosal spread of excitation from one sensorimotor cortex to the opposite cerebral cortex. The relative latencies of muscles on the same side of the body reflected not only the differences in efferent delays from the motor cortex via spinal cord and peripheral nerves, but also delays due to the spread of myoclonic activity within the sensorimotor cortex itself. This intrahemispheric spread followed a grossly somatotopic pattern. It is suggested that this spread involves cortico-cortical pathways. The additional delay due to spread of activity from hand to leg area of the sensorimotor cortex was about 10 ms in the first active hemisphere, in both generalized reflex and action jerks. The delays due to somatotopic spread of activity in the opposite later activated hemisphere were shorter. The tendency for spread of excitation through callosal and cortico-cortical pathways is an additional pathophysiological abnormality in some patients with cortical myoclonus, and may be important in the generalization of seizures seen in these patients.

Adult↗
PubMed2004

Positive resection margins in partial penectomies: sites of involvement and proposal of local routes of spread of penile squamous cell carcinoma.

Recurrence in patients with penile carcinoma occurs in about one third of cases, usually due to insufficient surgery or positive resection margins. An evaluation of surgical resection margins in penectomy specimens was performed to determine precise anatomic sites of tumor involvement, hoping to advance knowledge concerning the local routes of spread of penile carcinomas. A pathologic study of 80 partial penectomies revealed 14 positive margins. Margins were examined after their separation from the main specimen as follows: 1) proximal urethra and surrounding tissues consisting of urethral epithelium with Litree glands, lamina propria, corpus spongiosum, and penile fascia (periurethral cylinder); 2) proximal shaft with corresponding corpora cavernosa separated and surrounded by the tunica albuginea and penile fascia; and 3) skin of shaft with underlying corporal dartos. In 9 patients, only one site was involved by carcinoma, and in 5 there were multiple contiguous sites (for a total of 20 anatomic sites). The distribution of the various sites involved by carcinoma was as follows: urethral epithelium, 4 cases (2 in situ and 2 invasive carcinomas including intraluminal spread); lamina propria, 5 cases; corpus spongiosum, 3 cases; penile fascia, 6 cases; and corpora cavernosa and skin, 1 case each. One of the in situ lesions was discontinuous with the main glans tumor, and the other one was continuous with it. The penile fascia was the most commonly involved site followed by the urethral lamina propria and epithelium. Dissemination to outer skin, corpora cavernosa, and corpus spongiosum was less frequent. The highly vascularized and innervated loose connective tissue of the penile fascia appears to facilitate tumor spread. The urethra is either a pathway for in situ tumor progression from glans to urethra or part of a field prone to malignant transformation. The infrequent involvement of corpora cavernosa is probably due to the tunica albuginea acting as a barrier preventing tumor spread. Based on these observations and the examination of hundreds of penectomy specimens, we are proposing five probable routes of local spread for penile cancer: 1) horizontal and superficially spreading from one epithelial mucosal compartment (glans, coronal sulcus, and foreskin) to the other; 2) following the penile fascia; 3) through spaces created by feeding vessels in the tunica albuginea; 4) vertical spreading involving step-by-step different penile anatomic compartments; and 5) along the urethral epithelium.

Carcinoma, Squamous Cell↗
PubMed2001

Celiac plexus block: injectate spread and pain relief in patients with regional anatomic distortions.

BACKGROUND: The success of the neurolytic celiac plexus block, despite different approaches and methods used, depends on adequate spread of the injectate in the celiac area. This retrospective study was conducted to evaluate the patterns of alcohol spread and pain relief in patients with cancer or therapy-related anatomic distortion of the celiac area. METHODS: From 177 cancer patients who underwent computed tomography (CT)-guided single-needle neurolytic celiac plexus block via an anterior approach, a radiologist, blind to the aim of the study, retrospectively selected 105 patients with abnormal anatomy of the celiac area as judged by CT images obtained before the block. To evaluate CT patterns of neurolytic (mixed with contrast) spread, the celiac area was divided on the frontal plane into four quadrants: upper right and left and lower right and left, as related to the celiac artery. Results were expressed as the number of quadrants into which contrast spread, ie., four, three, two, or one quadrants with contrast. The patterns of contrast spread according to the number of quadrants with anatomic distortion were analyzed. Patient assessment by visual analog scale was reviewed to evaluate the degree of pain relief. Pain relief 30 days after block was considered long-lasting. Pain relief at 30 days after block was analyzed according to the number of quadrants with contrast. RESULTS: Overall, four, three, two, and one quadrants with contrast were observed in 9 (8%), 21 (20%), 49 (47%), and 26 (25%) patients, respectively. An inverse correlation was observed between the number of quadrants with anatomic distortion and the number of quadrants with contrast (P < 0.001). Long-lasting pain relief was noticed in nine of nine patients (100%; 95% confidence interval, 66-100) with contrast in four-quadrants, and in 10 of 21 patients (48%; 95% confidence interval, 26-70) with contrast in 3 quadrants (P < 0.01). None of the 75 patients with contrast in two quadrants or one quadrant experienced long-lasting pain relief. CONCLUSIONS: These findings suggest that, using the single-needle anterior approach, the neurolytic spread in the celiac area is highly hampered by the regional anatomic alterations. It also appears that only a complete (four quadrants) neurolytic spread in the celiac area can guarantee long-lasting analgesia, and that this picture may be obtained in a very limited fraction of patients with regional anatomic alterations.

Adult↗
PubMed1999

Advanced concepts in the imaging of perineural spread of tumor to the trigeminal nerve.

Perineural spread of head and neck tumor represents extension of the primary tumor along the perineurium. Diagnosis of perineural spread of carcinoma often is delayed unless the clinician maintains a high index of suspicion. It may be insidious, and patients may be asymptomatic for years. Perineural spread of carcinoma has been associated with a poor prognosis; however, it is becoming increasingly realized that cure is possible in some cases, if the full extent of the disease is known and treated. Magnetic resonance imaging (MRI) can detect perineural spread of head and neck carcinoma and define its extent. MRI is the imaging modality of choice to assess perineural disease due to its superior tissue contrast and multiplanar capability. Perineural spread of head and neck carcinomas most commonly involves the trigeminal nerve. Obliteration of the fat within (a) the superior medial orbital (ophthalmic division territory), (b) the periantral fat plane (maxillary division distribution), and (c) the pterygopalatine fossa all are useful indicators of distal perineural disease. A perineural vascular plexus (PNVP) surrounds the trigeminal ganglion and proximal portions of the trigeminal nerve divisions. The trigeminal ganglion and proximal portions of its divisions usually are seen as discrete nonenhancing structures separate from the PNVP. Occasionally, isolated enhancement of the ganglion and the proximal portions of the maxillary and mandibular divisions as they exit the skull base may be seen. This may be an artifact related to head position or may represent avid enhancement of the PNVP. In these situations, evaluating all branches, and the entire course, of the trigeminal nerve for perineural spread can aid in determining whether the apparent enhancement is an isolated normal variant or represents nerve pathology such as perineural spread.

Carcinoma, Squamous Cell↗
PubMed2005

Early esophageal cancer: pattern of lymphatic spread and prognostic factors for long-term survival after surgical resection.

OBJECTIVE: The objective of this study was to assess the prevalence and pattern of lymphatic spread in patients with early squamous cell and adenocarcinoma and identify prognostic factors for long-term survival after resection and lymphadenectomy. SUMMARY BACKGROUND DATA: Limited endoscopic approaches without lymphadenectomy are increasingly applied in patients with early esophageal cancer. MATERIAL AND METHODS: A total of 290 patients with early esophageal cancer (157 adenocarcinoma, 133 squamous cell cancer) had surgical resection with systematic lymphadenectomy. Specimens were assessed for prevalence and pattern of lymphatic spread. Prognostic factors were determined by multivariate analysis. RESULTS: None of the 70 patients with adenocarcinoma limited to themucosa had lymphatic spread, as compared with 2 of 26 with mucosal squamous cell cancer. Lymphatic spread was more common in patients with submucosal squamous cell cancer as compared with submucosal adenocarcinoma (36.4% versus 20.7%). Although lymph node metastases were usually limited to locoregional lymph node stations in early adenocarcinoma, distant lymphatic spread was frequent in early squamous cell cancer. On multivariate analysis, only histologic tumor type and the presence of lymph node metastases were independent predictors of long-term survival. Five-year survival rate was 83.4% for early adenocarcinoma versus 62.9% for early squamous cell cancer and 48.2% versus 79.5% for patients with/without lymphatic spread. DISCUSSION: Prevalence and pattern of lymphatic spread as well as long-term prognosis differ markedly between early esophageal squamous cell and adenocarcinoma. Limited resection techniques and individualized lymphadenectomy strategies appear applicable in patients with early adenocarcinoma.

Adenocarcinoma↗
PubMed1993

Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin.

In a previous study, we reported that two synthetic peptides derived from the 33-kD carboxyl terminal cell/heparin-binding fragment of fibronectin A chain promoted keratinocyte adhesion but not spreading. Because keratinocytes are capable of spreading on the 33/66-kD fragments, we focused on identifying additional chemically synthesized peptides from the cell/heparin-binding fragments of fibronectin that might promote cell spreading. When plastic substrata were coated with peptide FN-C/H-V (WQPPRARI), which is derived from the carboxyl-terminal heparin-binding domain of all plasma fibronectin isoforms, keratinocytes adhered and displayed a spread morphology. In solution, soluble peptide FN-C/H-V inhibited cell spreading on intact fibronectin and on the 33/66-kD fragments. Furthermore, polyclonal antibodies raised against peptide FN-C/H-V also inhibited keratinocyte spreading on fibronectin and the 33/66-kD fragments. These data support the hypothesis that keratinocyte cell adhesion and cell spreading on fibronectin are mediated by multiple distinct domains and different regulatory processes.

Amino Acid Sequence↗
PubMed1980

Fibronectin (LETS) does not support epithelial cell spreading.

The dependence of epithelial cell spreading on fibronectin-containing media has been investigated, using purified human plasma fibronectin, and two in vitro assays: (1) epidermal outgrowths from measured pieces of mouse ear skin, and (2) spreading of dissociated cells from trypsinized guinea pig epidermis. Using these systems, appreciable spread occurred in media containing whole plasma and plasma deficient in fibronectin but no significant spread occurred in media enriched only in fibronectin. Returning fibronectin to deficient plasma did not enhance or reduce optimal cell spreading. These studies suggest that the factor(s) in human plasma which supports epithelial cell spreading in vitro is not fibronectin and that the chemistry of epithelial and fibroblast cell spreading is different.

Animals↗
PubMed2007

Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread.

We investigated the role of listeriolysin O (LLO) and the bacterial phospholipases PI-PLC and PC-PLC in cell-to-cell spread of Listeria monocytogenes. We showed that LLO is essential for cell-to-cell spread in primary murine macrophages. Electron micrographs revealed that in the absence of continued LLO expression, bacteria remain trapped in secondary spreading vacuoles having either a double or single membrane. In bacteria lacking PI-PLC and PC-PLC, cessation of LLO expression after initiation of infection resulted in a significant increase in the proportion of bacteria trapped in double-membrane compartments. We propose that the bacterial phospholipases are involved in the dissolution of the inner membrane of the spreading vacuole, yet are not sufficient for disruption of the outer membrane. As a consequence, we identified LLO as a key factor in the disruption of the outer membrane. This model is consistent with the observation that LLO is dispensable for cell-to-cell spread from human macrophages into a cell type in which LLO is not required for vacuolar escape. These data suggest that during human infection, spreading of L. monocytogenes to distant organs is likely to occur even in the absence of LLO expression, and that the bacterial phospholipases may be sufficient to mediate continued cell-to-cell spread.

Bacterial Toxins↗
PubMed1998

The functional significance of epitope spreading and its regulation by co-stimulatory molecules.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become major targets of an ongoing immune response. This phenomenon has been defined in experimental and natural situations as a consequence of acute or persistent infection and secondary to chronic tissue destruction that occurs during progressive autoimmune disease. We have investigated the functional significance of this process in the chronic stages of both autoimmune and virus-induced central nervous system (CNS) demyelinating disease models in the SJL/J mouse. During the relapsing-remitting course of experimental autoimmune encephalomyelitis (R-EAE) induced with defined encephalitogenic myelin peptides, CD4+ T cells specific for endogenous epitopes on both the initiating myelin protein (intramolecular epitope spreading) and distinct myelin proteins (intermolecular epitope spreading) are primed secondary to myelin destruction during acute disease and play a major functional role in mediating disease relapses. Similarly, epitope spreading to endogenous myelin epitopes appears to play a major functional role in the chronic-progressive course of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a virus-induced CD4+ T-cell-mediated immunopathology. In TMEV-IDD, myelin destruction is initiated by virus-specific CD4+ T cells which target virus epitopes persisting in CNS-derived antigen-presenting cells. However, the chronic stage of this progressive disease is associated with the activation of CD4+ T cells specific for multiple myelin epitopes. In both models, the temporal course of T-cell activation occurs in a hierarchical order of epitope dominance, spreading first to the most immunodominant epitope and progressing to lesser immunodominant epitopes. In addition, epitope spreading in R-EAE is regulated predominantly by CD28/B7-1 co-stimulatory interactions, as antagonism of B7-1-mediated co-stimulation using anti-B7-1 F(ab) fragments is an effective ameliorative therapy for ongoing disease. The process of epitope spreading has obvious important implications for the design of antigen-specific therapies for the treatment of autoimmune disease since these therapies will have to identify and target endogenous self epitopes associated with chronic tissue destruction.

Animals↗
PubMed1994

Role of beta 1 integrins in cell spreading and migration of human nevomelanocytes and dysplastic nevi cells on collagen type IV and laminin.

We characterized beta 1 integrin subunit expression on three different cultures of benign human nevomelanocytes (NMC) and on four different cell cultures of human dysplastic nevus (DN) cells by flow cytometry analysis and examined their role in mediating cell spreading and migration on collagen type IV (CN IV) and laminin (LN) coated substrates by using a quantitative video image analysis system. The seven human NMC and DNC cultures expressed heterogeneous levels of beta 1, alpha 2, alpha 3 and alpha 6 integrin subunits. Image analysis showed that a significant increase (P < 0.001) in cell spreading and migration of the DN cells was induced on increasing coating concentrations of CN IV and LN. However, the NMC did not show an increase in cell spreading or migration on these substrates when compared to the substrates coated with denatured BSA only. The CN IV-induced cell spreading of the DN cells was significantly inhibited by anti-beta 1 mAb (AIIB2), anti-alpha 2 mAb (P1E6), or anti-alpha 3 mAb (P1B5), but not by mAb against alpha 6 integrin subunit (GoH3). The DN cell spreading on LN was not significantly inhibited by these mAbs. In contrast, the migration of the DN on CN IV and LN was significantly inhibited by anti-beta 1 mAb, anti-alpha 2 mAb, anti-alpha 3 mAb and anti-alpha 6 mAb. These data suggest that the alpha 2 and alpha 3 subunit are important for cell spreading of the DN on CN IV, although they are less important in cell spreading on the extracellular matrix component LN.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1994

Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblasts spreading on titanium surfaces.

The adhesion and spreading of human gingival fibroblasts on glass and differently processed titanium surfaces was studied by immunolocalization of vinculin and the alpha and beta subunits of the fibronectin (alpha 5 beta 1) and (alpha v beta 3) receptors. Vinculin-containing focal contacts were present both at 4 and 24 h of spreading in cells grown on glass or electropolished or etched titanium surfaces but not in cells spreading on sandblasted titanium surfaces. Immunostaining for the alpha 5 and beta 1 subunits of the fibronectin receptor showed only a diffuse membrane fluorescence after 4 h of cell spreading irrespective of the growth surface. The alpha v and beta 3 subunits of the vitronectin receptor were at this stage detected in focal contacts in cells spreading on glass or electropolished or etched titanium surfaces. In cells spreading on sandblasted titanium surfaces, however, the vitronectin receptor had only a diffuse distribution. In cells that had been allowed to spread for 24 h on glass or electropolished or etched titanium surfaces the alpha 5 and beta 1 integrin subunits were either diffusely distributed or showed a localization typical of extracellular matrix contacts. The alpha v and beta 3 integrin subunits were, as earlier, localized to typical focal contacts in cells grown on glass or electropolished or etched titanium surfaces. Cells attached to sandblasted titanium surfaces still expressed all the integrin subunits only diffusely. The results show that the surface texture of the substratum can affect the expression of integrin subunits in human gingival fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1984

Genetic transformation in Streptococcus sanguis. Simultaneous variation of surface-spreading, competence, hemagglutination and polar fimbriation in selected strains.

Strains of Streptococcus sanguis expressing spreading zones around the colonies and competence in genetic transformation, were subcultivated in the laboratory, and in three strains a variation to non-spreading colony morphology on human blood agar plates was observed. These variants were isolated and compared with the original strains by genetic transformation, hemagglutination and electron microscopy. In genetic transformation with streptomycin resistance as genetic marker, the non-spreading variants showed a decrease to 1/50 or less of the transformation frequencies compared to the wild types. Hemagglutination of guinea-pig erythrocytes is a common characteristic of S.sanguis strains, but the non-spreading variants did not hemagglutinate. In electron microscopy studies the wild-type strains of S.sanguis, but not the non-spreading variants, presented long, polar fimbriae. One of the non-spreading variants had short, peritrichous fimbriae. The co-variation in surface-spreading, competence in genetic transformation, hemagglutination and polar fimbriation is discussed.

Fimbriae, Bacterial↗
PubMed2005

Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis.

Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K(+)) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K(+) requirement, directed mutagenesis of the higher-affinity K(+) transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K(+)-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K(+)-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K(+) ions, as well as growth conditions that promote sliding motility.

Acetoin↗
PubMed1999

Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence.

The pseudorabies virus (PRV) gE gene encodes a multifunctional membrane protein found in infected cell membranes and in the virion envelope. Deletion of the gE gene results in marked attenuation of the virus in almost every animal species tested that is permissive for PRV. A common inference is that gE mutants are less virulent because they have reduced ability to spread from cell to cell; e.g., gE mutants infect fewer cells and, accordingly, animals live longer. In this report, we demonstrate that this inference does not hold in a rat experimental model for virus invasion of the brain. We find that animals infected with gE mutants live longer despite extensive retrograde, transneuronal spread of virus in the rat brain. In this model of brain infection, virus is injected into the stomach musculature and virions spread to the brain in long axons of brain stem neurons that give rise to the tenth cranial nerve (the vagus). The infection then spreads from neuron to neuron in well-defined, and physically separated, areas of the brain involved in autonomic regulation of the viscera. We examined the progression of infection of five PRV strains in this circuitry: the wild-type PRV-Becker strain, the attenuated PRV-Bartha vaccine strain, and three gE mutants isogenic with the PRV-Becker strain. By 60 to 67 h after infection, all PRV-Becker-infected animals were dead. Analysis of Becker-infected rats killed prior to virus-induced death demonstrated that the virus had established an infection only in the primary vagal neurons connected directly to the stomach and synaptically linked neurons in the immediate vicinity of the caudal brain stem. There was little spread to other neurons in the vagus circuitry. In contrast, rats infected with PRV-Bartha or PRV-Becker gE mutants survived to at least 96 h and exhibited few overt signs of disease. Despite this long survival and the lack of symptoms, brains of animals sacrificed at this time revealed extensive transsynaptic infection not only of the brain stem but also of areas of the forebrain synaptically linked to neurons in the brain stem. This finding provides evidence that the gE protein plays a role in promoting symptoms of infection and death in animals that is independent of neuron-to-neuron spread during brain infection. When this early virulence function is not active, animals live longer, resulting in more extensive spread of virus in the brain.

Animals↗
PubMed2001

Herpes simplex virus with highly reduced gD levels can efficiently enter and spread between human keratinocytes.

The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

Cell Line↗
PubMed2002

Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse.

We used the neonatal mouse model of rotavirus infection to study extraintestinal spread following oral inoculation. Five-day-old pups were inoculated with either SA11-Cl3, SA11-Cl4, SA11-4F, RRV, or B223. By using virus detection in the liver as a proxy determination for extraintestinal spread, rotavirus strains capable of extraintestinal spread at high frequency (rhesus rotavirus [RRV]) and very low frequency (SA11-Cl4) were identified. Both strains productively infected the gastrointestinal tract. Oral inoculation of mice with RRV/ SA11-Cl4 reassortants and determination of virus titers in the gut and liver revealed that the extraintestinal spread phenotype segregated with RRV genome segment 7 to a high level of significance (P = 10(-3)). RRV segment 7 also segregated with the growth of virus in the gut (P = 10(-5)). Although infection of the gut was clearly required for tropism to the liver, there was no correlation between virus titers in the gut and detection of virus in the liver. Five days after intraperitoneal administration to bypass the gut barrier to virus spread, RRV and SA11-Cl4 both were recovered in the liver. However, only RRV was found in the liver following subcutaneous inoculation, suggesting that this peripheral site presented a similar barrier to virus spread as the gut. Sequence analysis of segment 7 from parental RRV and SA11-Cl4 and selected reassortants showed that (i) amino acid differences were distributed throughout the coding sequences and not concentrated in any particular functional motif and (ii) parental sequence was preserved in reassortants. These data support the hypothesis that NSP3, coded for by genome segment 7, plays a significant role in viral growth in the gut and spread to peripheral sites. The mechanism of NSP3-mediated tropism is under investigation.

Administration, Oral↗