ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

Distinct Cholesterol Localization in Glioblastoma Multiforme Revealed by Mass Spectrometry Imaging.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults and is highly resistant to chemo- and radiotherapies. GBM has been associated with alterations in lipid contents, but lipid metabolism reprogramming in tumor cells is not fully elucidated. One of the key hurdles is to localize the lipid species that are correlated with tumor growth and invasion. A better understanding of the localization of abnormal lipid metabolism and its vulnerabilities may open up to novel therapeutic approaches. Here, we use time-of-flight secondary ion mass spectrometry (ToF-SIMS) to spatially probe the lipid composition in a GBM biopsy from two regions with different histopathologies: one region with most cells of uniform size and shape, the homogeneous part, and the other with cells showing a great variation in size and shape, the heterogeneous part. Our results reveal elevated levels of cholesterol, diacylglycerols, and some phosphatidylethanolamine in the homogeneous part, while the heterogeneous part was dominated by a variety of fatty acids, phosphatidylcholine, and phosphatidylinositol species. We also observed a high expression of cholesterol in the homogeneous tumor region to be associated with large cells but not with macrophages. Our findings suggest that ToF-SIMS can distinguish in lipid distribution between parts within a human GBM tumor, which can be linked to different molecular mechanisms.

Involvement of GABAA receptors containing α6 subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging.

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.

Multimodal chemical imaging of a single brain tissue section using ToF-SIMS, MALDI-ToF and immuno/histochemical staining.

Cluster ion beam ToF-SIMS and/or MALDI-ToF mass spectrometry imaging (using 1,5-DAN matrix via sublimation) of a single coronal rat brain tissue section followed by classical- or immuno- histochemical staining faclilated a new multimodal chemical imaging workflow allowing complementary correlation of the lipid molecular ion images with the immuno/histological features within cerebellum region of the same brain tisue section.

Dark-field microscopy enhance visibility of CD31 endothelial staining.

A simple dark field microscopy technique was used for visualization of blood vessels in normal human renal tissues and carcinoma. Phase contrast condenser ring apt for high power objectives was combined with a 10x objective in order to create a dark field illumination of the specimens examined. The endothelial lining of the vessels had been stained by using CD31 monoclonal antibodies combined with conventional peroxidase immunohistochemistry. The final DAB addition used for this technique induced an intense light scatter in the dark field microscope. This scattered light originating from the endothelial lining made the walls of the bright vessels easily detectable from the dark background.

Elevated intracranial pressure after head trauma can be suppressed by antisecretory factor-a pilot study.

BACKGROUND: Control of intracranial pressure (ICP) is a key element in neurointensive care for directing treatment decisions in patients with severe traumatic brain injury (TBI). The anti-inflammatory protein antisecretory factor (AF) has been demonstrated to reduce experimentally induced high ICP in animal models. This report describes the first steps to investigate the uptake, safety, and influence of AF for reduction of elevated ICP in patients with TBI in a clinical setting. METHOD: Four patients with severe TBI (Glasgow Coma Scale < 9) that required neurointensive care with ICP monitoring due to signs of refractory intracranial hypertension were investigated. One hundred milliliters of Salovum®, a commercially available egg yolk powder with high contents of AF peptides, was administrated either via nasogastric (patients 1 and 2) or rectal tube (patients 2, 3, and 4) every 8 h for 2 to 3 days as a supplement to the conventional neurointensive care. ICP was registered continuously. Plasma levels of AF were measured by enzyme-linked immunosorbent assay (ELISA) to confirm that Salovum® was absorbed appropriately into the bloodstream. RESULTS: In the first two patients, we observed that when delivered by the nasogastric route, there was an accumulation of the Salovum® solution in the stomach with difficulties to control ICP due to impaired gastric emptying. Therefore, we tested to administer Salovum® rectally. In the third and fourth patients, who both showed radiological signs of extensive brain edema, ICP could be controlled during the course of rectal administration of Salovum®. The ICP reduction was statistically significant and was accompanied by an increase in blood levels of AF. No adverse events that could be attributed to AF treatment or the rectal approach for Salovum® administration were observed. CONCLUSIONS: The outcomes suggest that AF can act as a suppressor of high ICP induced by traumatic brain edema. Use of AF may offer a new therapeutic option for targeting cerebral edema in clinical practice.

Brain region-specific amyloid plaque-associated myelin lipid loss, APOE deposition and disruption of the myelin sheath in familial Alzheimer's disease mice.

There is emerging evidence that amyloid beta (Aß) aggregates forming neuritic plaques lead to impairment of the lipid-rich myelin sheath and glia. In this study, we examined focal myelin lipid alterations and the disruption of the myelin sheath associated with amyloid plaques in a widely used familial Alzheimer's disease (AD) mouse model; 5xFAD. This AD mouse model has Aß42 peptide-rich plaque deposition in the brain parenchyma. Matrix-assisted laser desorption/ionization imaging mass spectrometry of coronal brain tissue sections revealed focal Aß plaque-associated depletion of multiple myelin-associated lipid species including sulfatides, galactosylceramides, and specific plasmalogen phopshatidylethanolamines in the hippocampus, cortex, and on the edges of corpus callosum. Certain phosphatidylcholines abundant in myelin were also depleted in amyloid plaques on the edges of corpus callosum. Further, lysophosphatidylethanolamines and lysophosphatidylcholines, implicated in neuroinflammation, were found to accumulate in amyloid plaques. Double staining of the consecutive sections with fluoromyelin and amyloid-specific antibody revealed amyloid plaque-associated myelin sheath disruption on the edges of the corpus callosum which is specifically correlated with plaque-associated myelin lipid loss only in this region. Further, apolipoprotein E, which is implicated in depletion of sulfatides in AD brain, is deposited in all the Aß plaques which suggest apolipoprotein E might mediate sulfatide depletion as a consequence of an immune response to Aß deposition. This high-spatial resolution matrix-assisted laser desorption/ionization imaging mass spectrometry study in combination with (immuno) fluorescence staining of 5xFAD mouse brain provides new understanding of morphological, molecular and immune signatures of Aß plaque pathology-associated myelin lipid loss and myelin degeneration in a brain region-specific manner. Read the Editorial Highlight for this article on page 7.

Antisecretory factor AF-16 improves vascular access to a rat mammary tumour.

Tumor tissue often has an insufficient nutritional supply, in part due to compression of the vascular network from an increased interstitial fluid pressure. We have shown that the antisecretory factor peptide AF-16 can reduce this pressure in experimental rat breast tumors. In this work we studied if AF-16 administration opened up to an increased vascular volume in these tumors. Sprague-Dawley rats were given dimethylbenxanthracene and developed mammary tumors which were studied. Evans Blue was used as an intravascular volume indicator. Under anesthesia the rats were given AF-16 or solvent intranasally, and Evans Blue was injected i.v. 45 min later. Tumors and various organs were dissected and Evans Blue was extracted and colorimetrically quantified. Tumors had a significantly higher vascular volume after AF-16 administration as compared to other organs. Liver and renal vascular volumes were also increased but to a lesser degree than in the tumors. The results indicate that AF16 could be a candidate for increasing vascular access for chemotherapy in cancer therapy.

Spatial Lipidomics Reveals Region and Long Chain Base Specific Accumulations of Monosialogangliosides in Amyloid Plaques in Familial Alzheimer's Disease Mice (5xFAD) Brain.

Ganglioside metabolism is significantly altered in Alzheimer's disease (AD), which is a progressive neurodegenerative disease prominently characterized by one of its pathological hallmarks, amyloid deposits or "senile plaques". While the plaques mainly consist of aggregated variants of amyloid-ß protein (Aß), recent studies have revealed a number of lipid species including gangliosides in amyloid plaques along with Aß peptides. It has been widely suggested that long chain (sphingosine) base (LCBs), C18:1-LCB and C20:1-LCB, containing gangliosides might play different roles in neuronal function in vivo. In order to elucidate region-specific aspects of amyloid-plaque associated C18:1-LCB and C20:1-LCB ganglioside accumulations, high spatial resolution (10 µm per pixel) matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) of gangliosides in amyloid plaques was performed in hippocampal and adjacent cortical regions of 12 month old 5xFAD mouse coronal brain sections from two different stereotaxic coordinates (bregma points, -2.2 and -2.7 mm). MALDI-IMS uncovered brain-region (2 and 3D) and/or LCB specific accumulations of monosialogangliosides (GMs): GM1, GM2, and GM3 in the hippocampal and cortical amyloid plaques. The results reveal monosialogangliosides to be an important component of amyloid plaques and the accumulation of different gangliosides is region and LCB specific in 12 month old 5xFAD mouse brain. This is discussed in relation to amyloid-associated AD pathogenesis such as lipid related immune changes in amyloid plaques, AD specific ganglioside metabolism, and, notably, AD-associated impaired neurogenesis in the subgranular zone.

On-Tissue Chemical Derivatization of Catecholamines Using 4-( N-Methyl)pyridinium Boronic Acid for ToF-SIMS and LDI-ToF Mass Spectrometry Imaging.

The analysis of small polar compounds with ToF-SIMS and MALDI-ToF-MS have been generally hindered by low detection sensitivity, poor ionization efficiency, ion suppression, analyte in-source fragmentation, and background spectral interferences from either a MALDI matrix and/or endogenous tissue components. Chemical derivatization has been a well-established strategy for improved mass spectrometric detection of many small molecular weight endogenous compounds in tissues. Here, we present a devised strategy to selectively derivatize and sensitively detect catecholamines with both secondary ion ejection and laser desorption ionization strategies, which are used in many imaging mass spectrometry (IMS) experiments. Chemical derivatization of catecholamines was performed by a reaction with a synthesized permanent pyridinium-cation-containing boronic acid molecule, 4-( N-methyl)pyridinium boronic acid, through boronate ester formation (boronic acid-diol reaction). The derivatization facilitates their sensitive detection with ToF-SIMS and LDI-ToF mass spectrometric techniques. 4-( N-Methyl)pyridinium boronic acid worked as a reactive matrix for catecholamines with LDI and improved the sensitivity of detection for both SIMS and LDI, while the isotopic abundances of the boron atom reflect a unique isotopic pattern for derivatized catecholamines in MS analysis. Finally, the devised strategy was applied, as a proof of concept, for on-tissue chemical derivatization and GCIB-ToF-SIMS (down to 3 µm per pixel spatial resolution) and LDI-ToF mass spectrometry imaging of dopamine, epinephrine, and norepinephrine in porcine adrenal gland tissue sections. MS/MS using collision-induced dissociation (CID)-ToF-ToF-SIMS was subsequently employed on the same tissue sections after SIMS and LDI mass spectrometry imaging experiments, which provided tandem MS information for the validation of the derivatized catecholamines in situ. This methodology can be a powerful approach for the selective and sensitive ionization/detection and spatial localization of diol-containing molecules such as aminols, vic-diols, saccharides, and glycans along with catecholamines in tissue sections with both SIMS and LDI/MALDI-MS techniques.

Adipose tissue and body composition in women six years after gestational diabetes: factors associated with development of type 2 diabetes.

Factors differentiating women at highest risk of progression to type 2 diabetes mellitus (T2DM) after gestational diabetes mellitus (GDM) are incompletely known. Our aim was to characterize adipose tissue and body composition in relation to glucose metabolism in women with a history of GDM and to identify factors associated with development of T2DM. We examined glucose tolerance (OGTT), insulin sensitivity (HOMA-IR), body composition (anthropometry, air displacement plethysmography), and blood chemistry in 39 women 6 years after GDM. An adipose tissue biopsy was obtained to assess the size, number, and lipolytic activity of adipocytes, and adipokine release and density of immune cells and blood vessels in adipose tissue. Normal glucose tolerance (NGT) was identified in 31 women and impaired glucose metabolism (IGM) in 8. Women with IGM had higher BMI/fat mass, and related expected adipose tissue features, than women with NGT. Ethnicity was similar in the groups, but numerically there was a higher proportion of European women in the NGT group and a higher proportion of non-European women in the IGM group. BMI was the best discriminator of NGT versus IGM (multivariable logistic regression: OR = 1.34, P < 0.01). Waist-to-height ratio and adipocyte volume were most strongly associated with HOMA-IR (multivariable linear regression: R2 = 0.656, P < 0.001). After adjustment for BMI/ethnicity, women with IGM had increased serum adipocyte fatty acid-binding protein, weight gain after index pregnancy, and a lower proportion of fat-free mass. These factors, together with high BMI, abdominal fat distribution, and enlarged adipocytes, may increase the risk of progression to T2DM after GDM.

Free lipid and computerized determination of adipocyte size.

The size distribution of adipocytes in a suspension, after collagenase digestion of adipose tissue, can be determined by computerized image analysis. Free lipid, forming droplets, in such suspensions implicates a bias since droplets present in the images may be identified as adipocytes. This problem is not always adjusted for and some reports state that distinguishing droplets and cells is a considerable problem. In addition, if the droplets originate mainly from rupture of large adipocytes, as often described, this will also bias size analysis. We here confirm that our ordinary manual means of distinguishing droplets and adipocytes in the images ensure correct and rapid identification before exclusion of the droplets. Further, in our suspensions, prepared with focus on gentle handling of tissue and cells, we find no association between the amount of free lipid and mean adipocyte size or proportion of large adipocytes.

Low levels of anti-secretory factor in placenta are associated with preterm birth and inflammation.

INTRODUCTION: Anti-secretory factor is a protein that regulates secretory and inflammatory processes and preterm birth is associated with inflammation. Therefore, our hypothesis was that anti-secretory factor might play a role in immune reactivity and homeostasis during pregnancy. MATERIAL AND METHODS: Following spontaneous onset of labor and preterm or term delivery, placenta biopsies were collected. The levels of anti-secretory factor and markers of inflammation (CD68, CD163) and vascularization (CD34, smooth muscle actin) were analyzed by immunohistochemistry. RESULTS: The 61 placental biopsies included 31 preterm (<37 weeks of gestation) and 30 term (37-41 weeks) samples. The preterm placentas exhibited lower levels of anti-secretory factor (p = 0.008) and larger numbers of CD68-positive cells (p < 0.001) compared to term. Preterm placentas had blood vessel of smaller diameter (p = 0.036) indicative of immaturity. The level of interleukin-6 in cord blood was higher after very preterm than term birth, suggesting a fetal inflammatory response. The placenta level of anti-secretory factor was positively correlated to the length of gestation (p = 0.025) and negatively correlated to the levels of the inflammatory markers CD68 (p = 0.015) and CD163 (p = 0.028). CONCLUSIONS: Preterm delivery is associated with low levels of anti-secretory factor in placenta. Inflammation, a potential trigger of preterm birth, is more pronounced in the preterm placenta and inversely related to the placental level of anti-secretory factor, suggesting both a link and a potential target for intervention.

Dual polarity MALDI imaging mass spectrometry on the same pixel points reveals spatial lipid localizations at high-spatial resolutions in rat small intestine.

Sensitive laser desorption/ionization obtained via a sublimation-coated 1,5-diaminonaphthalene (1,5-DAN) matrix allowed dual polarity MALDI-IMS analysis on the same pixel points across the jejunal mucosal region in rat small intestine which yielded high-spatial-resolution (10 µm) ion images of several lipid species correlated with the same histological features.

TRPA1 Mechanoreceptors Mediate the IL-6 Response to a Single PD Dwell in the Rat.

BACKGROUND: The development of modern, biocompatible peritoneal dialysis (PD) fluids has not entirely eliminated the local pro-inflammatory effects of PD fluid administration. The present study was performed in order to establish the importance of known signaling pathways connected to mechano-, osmo- and chemo-sensors of the transient receptor potential (TRP) family for the acute inflammatory response to PD. METHODS: Rats were exposed to a single 4-hour dwell of lactate-buffered, 2.5% glucose, filter-sterilized PD fluid through an implanted PD catheter. In some groups, the PD dwell was preceded by intravenous administration of blockers of TRPV1 (BCTC), TRPA1 (HC030031), or neurokinin 1 (NK1) (Spantide II) receptors. Cytokine messenger ribonucleic acid (mRNA) expressions were quantified in tissue biopsies (real-time polymerase chain reaction [qPCR]), and cytokine concentrations were quantified in dialysate samples by enzyme-linked immunosorbent assay (ELISA). Tissue expressions of TRPV1, TRPA1, and NK1 were evaluated immuno-histochemically. RESULTS: The PD dwell induced peritoneal synthesis of Il1b, Tnf, and Il6 and a secretion of interleukin-6 (IL-6) into the dialysate. The catheter implantation already induced the transcription of Il1b and Tnf but did not significantly affect Il6 transcription. The Il6 response to the PD dwell could be virtually eliminated by blocking TRPA1 but was not affected by TRPV1 blockade. Blocking the substance P receptor, NK1, produced an insignificant trend towards Il6 inhibition. TRPA1 and NK1 showed a stronger immuno-reactivity than TRPV1 on cells of the peritoneal tissue. CONCLUSION: The results show that IL-6 synthesis and secretion were connected to acute PD fluid exposure, and this response was triggered by TRPA1 receptors, possibly located to non-neuronal cells.

Food-induced changes of lipids in rat neuronal tissue visualized by ToF-SIMS imaging.

Time of flight secondary ion mass spectrometry (ToF-SIMS) was used to image the lipid localization in brain tissue sections from rats fed specially processed cereals (SPC). An IonTof 5 instrument equipped with a Bi cluster ion gun was used to analyze the tissue sections. Data from 15 brain samples from control and cereal-fed rats were recorded and exported to principal components analysis (PCA). The data clearly show changes of certain lipids in the brain following cereal feeding. PCA score plots show a good separation in lipid distribution between the control and the SPC-fed group. The loadings plot reveal that the groups separated mainly due to changes in cholesterol, vitamin E and c18:2, c16:0 fatty acid distribution as well as some short chain monocarboxylic fatty acid compositions. These insights relate to the working mechanism of SPC as a dietary supplement. SPC is thought to activate antisecretory factor (AF), an endogenous protein with regulatory function for inflammation and fluid secretion. These data provide insights into lipid content in brain following SPC feeding and suggest a relation to activating AF.

Mass spectrometric profiling of lipids in intestinal tissue from rats fed cereals processed for medical conditions.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used for lipid profiling of intestine tissue sections from rats fed specially processed cereals and rats fed ordinary feed as a control. This cereal is known to increase the activity of antisecretory factor in plasma and the exact mechanism for the activation process at the cellular level is unclear. ToF-SIMS has been used to track food induced changes in lipid content in intestinal tissue sections to gain insight into the possible mechanisms involved. Data from 20 intestine sections belonging to four different rats from each group of control and specially processed cereals-fed rats were obtained using the stage scan macroraster with a lateral resolution of 5 µm. Data were subsequently subjected to orthogonal partial least squares discriminant analysis. The data clearly show that changes of certain lipids are induced by the specially processed cereal feed. Scores plots show a well-defined separation between the two groups. The corresponding loading plots reveal that the groups separate mainly due to changes of vitamin E, phosphocholine, and phosphosphingolipid fragments, and that for the c18:2 fatty acid. The observed changes in lipids might give insight into the working mechanisms of antisecretory factor in the body, and this has been successfully used to understand the working mechanism of specially processed cereal-induced antisecretory factor activation in intestine.

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles.

Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses.

Macrophage Phenotype Is Associated With the Regenerative Response in Experimental Replacement of the Porcine Esophagus.

A porcine model for bridging circumferential defects in the intrathoracic esophagus has been developed in order to improve the treatment of children born with long-gap esophageal atresia. The aim of this study was to identify factors beneficial for tissue regeneration in the bridging area in this model and to describe the histological progression 20 days after replacement with a silicone-stented Biodesign mesh. Resection of 3 cm of intrathoracic esophagus and replacement with a bridging graft was performed in six newly weaned piglets. They were fed through a gastrostomy for 10 days, and then had probe formula orally for another 10 days prior to sacrifice. Two out of six piglets had stent loss prior to sacrifice. In the four piglets with the stent in place, a tissue tube, with visible muscle in the wall, was seen at sacrifice. Histology showed that the wall of the healing area was well organized with layers of inflammatory cells, in-growing vessels, and smooth muscle cells. CD163+ macrophages was seen toward the esophageal lumen. In the animals where the stent was lost, the bridging area was narrow, and histology showed a less organized structure in the bridging area without the presence of CD163+ macrophages. This study indicates that regenerative healing was seen in the porcine esophagus 20 days after replacement of a part of the intrathoracic esophagus with a silicone-stented Biodesign mesh, if the bridging graft is retained. If the graft is lost, the inflammatory pattern changes with invasion of proinflammatory, M1 macrophages in the entire wall, which seems to redirect the healing process toward scar formation.

Novel rat models to study primary genital herpes simplex virus-2 infection.

In this study we describe that six rat models (SD, WIST, LEW, BN, F344 and DA) are susceptible to intravaginal herpes simplex virus-2 (HSV-2) infection after pre-treatment with progesterone. At a virus dose of 5 × 10(6) PFU of HSV-2, all rat models were infected presenting anti-HSV-2 antibodies, infectious virus in vaginal washes, and HSV-2 DNA genome copies in lumbosacral dorsal root ganglia and the spinal cord. Most of the LEW, BN, F344, and DA rats succumbed in systemic progressive symptoms at day 8-14 post infection, but presented no or mild genital inflammation while SD and WIST rats were mostly infected asymptomatically. Infected SD rats did not reactivate HSV-2 spontaneously or after cortisone treatment. In an HSV-2 virus dose reduction study, F344 rats were shown to be most susceptible. We also investigated whether an attenuated HSV-1 strain (KOS321) given intravaginally, could protect from a subsequent HSV-2 infection. All LEW, BN, and F344 rats survived a primary HSV-1 infection and no neuronal infection was established. In BN and F344 rats, anti-HSV-1 antibodies were readily detected while LEW rats were seronegative. In contrast to naïve LEW, BN, and F344 rats where only 3 of 18 animals survived 5 × 10(6) PFU of HSV-2, 23 of 25 previously HSV-1 infected rats survived a challenge with HSV-2. The described models provide a new approach to investigate protective effects of anti-viral microbicides and vaccine candidates, as well as to study asymptomatic primary genital HSV-2 infection.