Tumor necrosis factor-alpha in cisplatin nephrotoxicity: a homebred foe?

A robust inflammatory response involving tumor necrosis factor-alpha (TNF-alpha) is induced during cisplatin nephrotoxicity. Using chimeric models, Reeves and colleagues now demonstrate that resident kidney cells, rather than infiltrating immune cells, are the major producers of TNF-alpha. Blockade of TNF-alpha attenuates inflammation and associated kidney injury.

Acute retinal necrosis: insights into pathogenesis from the mouse model.

Acute retinal necrosis (ARN) is a relatively rare syndrome that is caused by infection with one of several members of the human herpesvirus family. ARN usually occurs in otherwise healthy patients, although it has also been observed in immunocompromised individuals. It is characterized by retinal vasculitis and haemorrhaging, areas of retinal necrosis, vitreous and aqueous inflammation and optic neuritis. It may affect one or both eyes and frequently results in severely reduced vision or blindness in the affected eye. Results using the mouse model of ARN have provided insight into the pathogenesis of this disease. However, many unanswered questions remain, such as why does only a very small fraction of individuals infected with one or more herpesvirus develop ARN? Increased understanding of the interactions of herpesviruses with T cells and cytokines may enable the development of therapeutic strategies targeted specifically to control viral infection in the eye and/or brain.

Murine cytomegalovirus infection causes apoptosis of uninfected retinal cells.

PURPOSE: To determine the role of apoptosis in prevention and/or exacerbation of retinal disease in a mouse model of cytomegalovirus retinitis. METHODS: Immunocompetent or T-cell- depleted BALB/c mice were injected with murine cytomegalovirus (MCMV) by supraciliary injection. On sequential days after infection, mice were killed, and eyes were harvested for cryosectioning or for DNA extraction. Ocular sections were stained with monoclonal antibodies specific for MCMV or for T cells or used in the TdT-dUTP terminal nick-end labeling (TUNEL) assay to detect apoptotic cells. RESULTS: In immunocompetent BALB/c mice, TUNEL assays revealed that a large area of the retina was apoptotic in relation to the relatively small number of MCMV-infected cells that were observed in the subjacent choroid and/or retinal pigment epithelium. In infected eyes from T-cell- depleted mice, there were more TUNEL-positive cells, and the areas of apoptosis were more extensive than in immunocompetent mice. These observations correlated with the increased extent of MCMV infection that is observed in the eyes of T-cell- depleted mice. However, irrespective of immune status, TUNEL-positive apoptotic cells were present mainly in areas of the retina overlying areas of MCMV-infected choroid and/or retinal pigment epithelium. More intense DNA laddering, indicative of increased apoptosis, was observed in the posterior segments of the eyes of T-cell- depleted mice after supraciliary inoculation with murine cytomegalovirus compared with less intense DNA laddering in the posterior segments of eyes of immunocompetent MCMV-infected mice. CONCLUSIONS: The ability of the mouse's immune system to control MCMV infections in some tissues depends on induction of apoptosis in virus-infected cells. However, in the retina, cells undergoing apoptosis were not virus-infected, a finding that suggests that apoptosis of uninfected retinal cells may play a role in the pathogenesis of MCMV retinitis.

Natural killer cells prevent direct anterior-to-posterior spread of herpes simplex virus type 1 in the eye.

PURPOSE: Anterior chamber (AC) inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1) results in morphologic sparing of the ipsilateral retina, whereas the retina of the uninoculated contralateral eye becomes infected and undergoes acute retinal necrosis. Natural killer (NK) cells are an important component of the primary immune response to most virus infections. The purpose of this study was to determine whether NK cells are involved in preventing early direct anterior-to-posterior spread of HSV-1 after AC inoculation. METHODS: Normal BALB/c mice were inoculated with 4 X 10(4) plaque-forming units (PFU) of the KOS strain of HSV-1 using the AC route. NK activity was measured in the spleen, the superficial cervical and submandibular lymph nodes, and the inoculated eye by lysis of chromium-labeled, NK-sensitive YAC-1 target cells. Histopathologic scoring and immunohistochemical staining for HSV-1 were performed in NK-depleted (injected intravenously with anti-asialo GM1) or mock-depleted (injected intravenously with normal rabbit serum) mice. RESULTS: In mock-depleted mice, NK activity in the spleens, superficial cervical and submandibular lymph nodes, and inoculated eyes peaked at postinoculation (pi) day 5 and declined by pi day 7. Treatment with anti-asialo GM1 eliminated NK activity in the eye and at nonocular sites. The histopathologic scores at pi day 5 indicated more damage to the retinas of NK-depleted mice than to those of mock-depleted mice, and immunohistochemical staining for HSV-1 showed spread of the virus to the sensory retina only in NK-depleted mice. CONCLUSIONS: NK cells were activated within 5 days after AC inoculation of the KOS strain of HSV-1. Activation of NK cells appears to play a role in preventing direct anterior-to-posterior spread of the virus in the inoculated eye which, in turn, protects the retina of this eye and helps to explain why the architecture of the retina of this eye is spared.

Protection against murine cytomegalovirus retinitis by adoptive transfer of virus-specific CD8+ T cells.

PURPOSE: Human cytomegalovirus retinitis, the most common ophthalmic infection of AIDS patients, has been modeled in BALB/c mice infected with murine cytomegalovirus by the supraciliary route. A series of depletion and adoptive transfer studies was performed to determine whether adoptive transfer of T cells protects mice from retinitis caused by murine cytomegalovirus infection after supraciliary inoculation and to determine which subset of T cells is responsible for protection. METHODS: BALB/c mice were thymectomized and T cell-depleted by injection of monoclonal antibodies to CD4, CD8, or both. Murine cytomegalovirus (9 x 10(2) plaque forming units [pfu]) was injected into the supraciliary space. Experimental animals received murine cytomegalovirus-specific T cells or subsets of T cells 2 hours before virus injection, whereas control animals received herpes simplex virus type 1-specific T cells by tail vein injection. Eight days after virus injection, retinal pathology was scored by histopathologic examination of hematoxylin and eosin-stained ocular sections. RESULTS: CD8+ T cell depletion was sufficient for development of retinitis after supraciliary injection of murine cytomegalovirus. Adoptive transfer of murine cytomegalovirus-specific T cells, but not herpes simplex virus type 1-specific T cells, provided protection from retinitis. Additionally, separation of the murine cytomegalovirus-specific T cells into CD8+ and CD4+ subsets before adoptive transfer showed that the CD8+ fraction of the adoptive T cells was responsible for protection. CONCLUSIONS: These results suggest that adoptive transfer of cytomegalovirus-specific T cells or T cell subsets might be used to treat or prevent cytomegalovirus retinitis in immunosuppressed human patients.

Is there a risk of cytomegalovirus transmission during in vitro fertilization with donated oocytes?

OBJECTIVE: To define the risk of human cytomegalovirus (HCMV) transmission from donated oocytes. DESIGN: Prospective study. SETTING: University IVF program. PATIENT(S): Sixty-seven couples undergoing 72 cycles of IVF-ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum from both partners (women: n = 71; men: n = 60) was obtained for detection of antibodies to HCMV. Semen before preparation (n = 53), sperm after preparation (Percoll gradient; n = 47), cervical mucus aspirated at the time of oocyte aspiration (n = 70), and uninseminated oocytes and embryos not suitable for cryopreservation (n = 568) were frozen in liquid nitrogen. Polymerase chain reaction was used for detection of HCMV (immediate early 1 gene) in all samples collected. RESULT(S): Serum antibodies to HCMV were found in 62% of the women and 37% of the men tested. Human cytomegalovirus DNA was detected in 25% of the ejaculates and in 19% of the cervical mucus samples. There was no amplification of HCMV DNA from oocytes or embryos. CONCLUSION(S): Because we were unable to amplify HCMV DNA from any of the oocytes or embryos, it seems unlikely that HCMV is transmissible through oocyte or embryo donation.

NK cell modulation of murine cytomegalovirus retinitis.

CMV retinitis, the most common ophthalmic infection of AIDS patients, causes blindness if left untreated. To study the role of NK cells in the modulation of CMV ocular infection, 9.0 x 10(2) plaque-forming units of the Smith strain of murine CMV (MCMV) was injected into the supraciliary space of the left eyes of BALB/c mice. Lysis of NK-sensitive target cells (YAC-1) by effectors from the draining lymph nodes peaked at day 5 postinfection, while the splenic cytolytic response was biphasic, with peaks at days 2 and 7 postinfection. Flow cytometry showed that NK cells (DX-5+) increased in spleens and eyes 5 days after supraciliary infection with MCMV compared with uninfected or mock-infected controls. Eight days after supraciliary injection with 9.0 x 10(2) plaque-forming units of MCMV, 7 of 10 NK-depleted mice developed retinitis compared with only 2 of 10 non-NK-depleted control mice. Poly(I-C) activation of NK cells in T cell-depleted animals protected mice from MCMV retinitis; only 2 of 10 mice in the poly(I-C)-treated group developed retinitis compared with 8 of 10 T cell-depleted, non-poly(I-C)-treated control mice. These results show the importance of NK cells in preventing MCMV retinitis and suggest that NK cells may also be involved in modulation of cytomegalovirus retinitis in human patients.

T cells in the uninjected eye after anterior chamber inoculation of herpes simplex virus type 1.

PURPOSE: To investigate T cell infiltration in the posterior segment of the uninjected eye after uniocular anterior chamber inoculation of HSV-1. METHODS: The anterior chamber of one eye of euthymic BALB/c mice was injected with 1 x 10(4) plaque-forming units (PFU) to 2 x 10(4) PFU of herpes simplex virus type 1 (HSV-1; KOS strain). All mice were examined for retinitis on day 8 postinoculation (p.i.). Only mice with retinitis were retained and used in these experiments. Animals were killed on days 9, 11, 14, 21, 35, and 63 p.i. The uninjected eyes were removed. Some of the uninjected eyes were sectioned and stained for CD4+ and CD8+ cells using the avidin-biotinylated enzyme complex method. Infiltrating cells were collected from the remaining uninoculated eyes and stained using rat anti-mouse monoclonal antibodies specific for CD4+ or CD8+ T cells, and the percentage of CD4+ and CD8+ T cells was determined by flow cytometry. RESULTS: At day 9 p.i. (acute retinitis), T cells were observed in the uvea but not in the retina of the contralateral eye. CD4+ and CD8+ cells were observed in the sensory retina coincident with the onset of retinal necrosis (day 11 p.i.), and CD4+ and CD8+ T cells continued to be detected in the remnants of the retina up to and including day 63 p.i. The maximum percentage of both CD4+ and CD8+ T cells was observed at day 21 p.i. CONCLUSIONS: These results demonstrate that T cells enter the retina of the uninoculated eye during HSV-1 infection. The observation that T cells arrive in the sensory retina at the onset of retinal necrosis and not during acute retinitis and the peak of virus replication provides further evidence that T cells play a role in development of retinal necrosis. The result that T cells are observed in the uninjected eye as late as day 63 p.i. suggests that T cells might also have a role in the resolution phase of the disease.

Spread of HSV-1 to the suprachiasmatic nuclei and retina in T cell depleted BALB/c mice.

Following uniocular anterior chamber inoculation of the KOS strain of HSV-1 in euthymic BALB/c mice, virus spreads from the injected eye to the brain, and from the brain to the optic nerve and retina of the uninjected eye by day 7 post inoculation (p.i.), but the optic nerve and retina of the injected eye are not infected with virus. Infection of the optic nerve and retina of the injected eye is observed only in athymic mice or in mice depleted of both CD4+ and CD8+ T cells. To determine the role of T cells in virus spread, adult female BALB/c mice were thymectomized and T cell depleted. Mice were co-injected with the KOS strain of HSV-1 and RH116, a thymidine kinase-negative mutant of KOS containing the Escherichia coli lac Z gene. Animals were sacrificed on days 3-7 p.i., and the eyes and brains were examined for blue-stained, virus-infected cells. A difference in the timing of virus infection was observed in the area of the suprachiasmatic nuclei only in mice depleted of both CD4+ and CD8+ T cells, and in this group, the contralateral suprachiasmatic nucleus was infected two days earlier. Since one route by which virus could infect the retina of the injected eye is via connections of the contralateral suprachiasmatic nucleus to the ipsilateral optic nerve, these findings suggest that (a) retinitis observed in the injected eyes of mice depleted of both CD4+ and CD8+ T cells results from virus infection of the contralateral suprachiasmatic nucleus followed by spread of virus to the ipsilateral optic nerve and retina and (b) early HSV-1 infection of the contralateral suprachiasmatic nucleus is prevented by a T cell dependent mechanism.

Immune effector cell (IEC)-mediated protection from HSV-1 retinitis occurs in the brain.

Following uniocular anterior chamber inoculation of the KOS strain of HSV-1 into euthymic BALB/c mice, virus spreads from the injected eye to the brain and from the brain to the optic nerve and retina of the uninjected eye resulting in retinitis. Adoptive transfer of HSV-1-specific immune effector cells (IEC) within 24 h of anterior chamber inoculation of virus prevents retinitis. To determine where protection occurs, mice were injected with HSV-1 via the anterior chamber route, and fluorescently-labeled HSV-1-specific-IEC or ovalbumin-specific-lymph node cells were adoptively transferred intravenously. The eyes and brains of these mice were sectioned and examined for virus-infected cells and for fluorescently-labeled adoptively transferred cells. None of the mice in the group receiving an adoptive transfer of virus-specific IEC had evidence of virus infection of the ipsilateral suprachiasmatic nucleus (SCN), whereas the ipsilateral SCN of all of the mice in the control groups were virus-positive by day 5 P.I. Since virus spreads from the ipsilateral SCN to the contralateral optic nerve and retina to cause retinitis in the uninoculated eye, the results of these studies suggest IEC-mediated protection from HSV-1 retinitis occurs proximal to the ipsilateral SCN. Furthermore, since only HSV-1-specific IEC conferred protection and only these cells were observed in the brain, protection and trafficking of cells after adoptive transfer was virus-specific.

Adoptive transfer of murine cytomegalovirus-immune lymph node cells prevents retinitis in T-cell-depleted mice.

PURPOSE: The purpose of this study was to determine whether adoptive transfer of murine cytomegalovirus (MCMV)-immune lymph node cells prevents retinitis in immunosuppressed mice. METHODS: Adult BALB/c mice were thymectomized and T-cell depleted using rat monoclonal antibodies specific for mouse CD4+ and CD8+ T-cells. The level of rat immunoglobulin G in the treated mice was monitored by enzyme-linked immunosorbent assay. Immune cells were labeled with PKH26-GH immediately before adoptive transfer, and flow cytometry was used to determine the percentage of adoptively transferred T-cells (PKH+, fluorescein isothiocyanate [FITC+]) in the spleens of the recipient mice 3 days after transfer. The ability of adoptively transferred cells to protect from retinitis was studied in T-cell-depleted mice injected with MCMV through the supraciliary route. Mice received 4 x 10(7) in vitro-restimulated MCMV-immune cells, 4 x 10(7) freshly isolated MCMV-immune cells, 4 x 10(7) freshly isolated ovalbumin-immune cells, or no cells (control group). RESULTS: The best time to balance depletion of endogenous T-cells with persistence of transferred cells was 3 weeks after T-cell depletion. Both restimulated and freshly isolated MCMV-immune cells conferred protection from retinitis. Freshly isolated ovalbumin-immune lymph node cells did not prevent retinitis, indicating that protection was virus-specific and merely was not because of transfer of antigen-activated lymph node cells. CONCLUSIONS: Adoptive immunotherapy has been used to prevent cytomegalovirus (CMV) infection in patients who have undergone transplantation, and, by extrapolation, the results of these studies suggest that adoptive immunotherapy with human CMV-specific immune cells might be used to prevent or ameliorate CMV retinitis in immunocompromised patients.

Spread of murine cytomegalovirus to inner ocular structures following disruption of the blood-retina barrier in immunosuppressed BALB/c mice.

PURPOSE: The aims of this study were to determine whether disruption of the blood-retina barrier (BRB) increases spread of murine cytomegalovirus (MCMV) to the eye after intraperitoneal inoculation and whether systemic immunosuppression influences the location of MCMV in the ocular compartment. METHODS: The BRB of the left eye of normal and immunosuppressed mice was disrupted by supraciliary inoculation of tissue culture medium followed 2 hours later by intraperitoneal injection of MCMV. Plaque assay of homogenized ocular tissue was used to determine the frequency of virus-positive eyes and the titer of virus in the eyes. Beta-galactosidase staining of frozen sections was used to locate virus in the eyes. RESULTS: In nonimmunosuppressed mice, the frequency of virus isolation, as well as the titer of virus, were significantly higher in eyes in which the BRB had been disrupted. Although the frequency of virus isolation was the same in both eyes of immunosuppressed mice, the titer of virus was significantly higher in the eye in which the BRB had been disrupted. The most striking result was that the location of virus was different in the nondisrupted eyes of immunosuppressed mice than it was in the disrupted eyes of immunosuppressed mice. In the former, virus was seen only in the outer ocular structures (conjunctiva, sclera, lacrimal gland), whereas in the latter, virus was observed in the retina and anterior segment (iris, ciliary body) as well as the outer ocular structures. CONCLUSIONS: The results of these studies suggest that ocular damage followed by increased spread of virus to and within the eye during systemic infection with CMV may be one mechanism by which development of CMV retinitis is facilitated in patients with acquired immune deficiency syndrome.

Immunosuppression induces transcription of murine cytomegalovirus glycoprotein H in the eye and at non-ocular sites.

In these studies, DNA PCR was used to identify sites of murine cytomegalovirus (MCMV) latency after inoculation of virus into the supraciliary space of the eye. Reverse transcription (RT) PCR for an immediate early gene and a late gene was used to identify putative sites of virus reactivation after methylprednisolone (steroid)-induced immunosuppression. Ten weeks after inoculation of 5 x 10(2) PFU of MCMV, BALB/c mice were immunosuppressed by intramuscular injection of steroid. Control mice were infected but not immunosuppressed. Two weeks after initiation of immunosuppression, mice were sacrificed. DNA and RNA extracted from homogenized tissues were amplified for immediate early gene 1 (IE1) and late gene, glycoprotein H (gH), DNA and mRNA by PCR and RT-PCR, respectively. Replicating virus was detected in homogenized ocular and non-ocular tissues by plaque assay. In the latently infected PBS-treated control group, viral DNA was detected in the inoculated eye and in several non-ocular tissues; IE1 mRNA was found in most of the DNA-positive tissues, while gH mRNA was amplified only in a few of the MCMV DNA-positive tissues from a single mouse. After immunosuppression, viral DNA and IE1 mRNA were detected at a higher frequency in various tissues of steroid-treated mice. gH mRNA was detected in a significantly higher number of the inoculated eyes, salivary glands and other non-ocular tissues of steroid-treated mice. After immunosuppression, low titers of infectious virus were recovered from the salivary glands of steroid-treated mice, but infectious virus was not recovered from the inoculated eye of either steroid-treated of non-immunosuppressed mice. The DNA PCR results suggest that after inoculation of 5 x 10(2) PFU of MCMV into the supraciliary space of euthymic BALB/c mice, virus becomes latent in the inoculated eye, salivary gland and other extraocular tissues. The RT-PCR results suggest that latent MCMV can be reactivated in multiple tissues by immunosuppression.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

T lymphocyte infiltration in the brain following anterior chamber inoculation of HSV-1.

Following inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1) into one anterior chamber of euthymic BALB/c mice, virus spreads from the injected eye to the central nervous system and from the central nervous system to the optic nerve and retina of only the uninoculated eye. In contrast, in athymic BALB/c mice or mice depleted of both CD4+ and CD8+ T cells, virus spreads to the optic nerve and retina of both the injected eye and the uninjected eye. To determine the location in the central nervous system where spread of virus to the optic nerve and retina of the injected eye is prevented, euthymic BALB/c mice were injected with a mixture of KOS and RH116, a mutant of KOS that contains the Escherichia coli beta-galactosidase (beta-gal) gene. Several animals were sacrificed each day; serial frozen sections of the brain were prepared and sequential sections were stained for beta-gal or for T cells. At all sites except the suprachiasmatic nuclei, virus and T cells arrived at approximately the same time. However, at day 5 post inoculation (PI), T cells were present in both the ipsilateral and the contralateral suprachiasmatic nuclei, but only the ipsilateral suprachiasmatic nucleus was virus-positive. Since virus spreads from the ipsilateral suprachiasmatic nucleus to the contralateral optic nerve, these results suggest that T cells infiltrating the area of the contralateral suprachiasmatic nucleus prior to the arrival of virus at this site prevent virus spread into the optic nerve of the inoculated eye.

Sjögren's syndrome: cytokine and Epstein-Barr viral gene expression within the conjunctival epithelium.

PURPOSE: In primary Sjögren's syndrome (SS), ocular surface changes within the conjunctival epithelium include lymphocytic infiltration, squamous cell metaplasia, and a reduction in goblet cell number. These changes may be the simple result of increased mechanical abrasion secondary to dryness. Alternatively, they may represent a local response to ocular and/or systemic inflammatory processes, perhaps in response to Epstein-Barr viral (EBV) infection, an agent recently implicated in the etiology of SS. To determine whether inflammatory processes or local infection by EBV contribute to the ocular surface pathology of SS, we examined the expression of inflammatory cell surface markers, cytokines, and EBV gene products within the ocular conjunctiva of patients with SS. METHODS: Ocular conjunctival tissue was isolated from patients with primary SS and nondry eye control patients by impression cytology or direct biopsy. These specimens were examined by immunofluorescence microscopy and reverse-transcriptase polymerase chain reaction (RT-PCR) for the expression of various markers. RESULTS: The authors found the frequency of expression of HLA-DR (P < 0.0001), ICAM-1 (P < 0.035), and IL-6 (P < 0.0001) to be significantly elevated in patients with primary SS versus nondry eye control patients. The IL-2 receptor and cytokines IL-1 beta and IL-8 were each found to be expressed with relatively high frequency in both patient populations, whereas mRNAs encoding cytokines IL-2, IFN-gamma, GM-CSF, and TGF-beta were not reproducibly detectable in either population. Messenger RNA encoding a marker for passive-latent EBV infection (EBNA-1) was detected with high frequency in both SS and normal populations. The EBV IL-10 analog BCRF-1 was expressed with low frequency in the SS population; however, these levels were not significantly different from the control population. The expression of two other markers of EBV infection, latent membrane protein (LMP, a lytic and latent marker), and BZLF-1 (putative latent-lytic switch gene) was undetectable in either study population. CONCLUSION: Based on the increased expression of the cell surface molecules HLA-DR and ICAM-1, and the inflammatory cytokine IL-6, the authors propose that local inflammatory processes contribute to the ocular surface changes and ocular surface dryness associated with primary SS.

Sparing of the ipsilateral retina after anterior chamber inoculation of HSV-1: requirement for either CD4+ or CD8+ T cells.

PURPOSE: To determine whether CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells are required for preservation of the ipsilateral retina after uniocular anterior chamber inoculation of herpes simplex virus type 1 (HSV-1). METHODS: Adult-thymectomized BALB/c mice were T cell depleted by administration of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, or anti-CD4 mAb and anti-CD8 mAb together. Control mice were thymectomized but were not T cell depleted. HSV-1 (KOS) was inoculated in one anterior chamber. At intervals after inoculation, the injected eyes were examined histopathologically or homogenized to determine the kinetics of infectious virus recovery. Additional groups of in vivo depleted mice were injected with wild type KOS and RH116 (a mutant of KOS containing the Escherichia coli beta-galactosidase gene) to determine whether viral genes were expressed in the retina in any of the mice. RESULTS: In the inoculated eyes of mice depleted of both CD4+ and CD8+ T cells, there was a significantly higher incidence of acute destructive retinitis at days 9 and 14 postinoculation (PI), and the titer of virus recovered at day 14 PI was significantly higher. Viral gene expression in the retina and the optic nerve was observed after day 7 PI only in the group of mice depleted of both CD4+ and CD8+ cells. In contrast, acute destructive retinitis was not observed in nondepleted mice or in mice depleted of either CD4+ or CD8+ T cells alone, and virus recovery was not significantly different among these three groups of mice. No virus-infected cells were observed in the optic nerve or the sensory retina of nondepleted mice, of mice depleted of only CD4+ cells, or of mice depleted of only CD8+ cells. CONCLUSION: The results of these studies suggest that either CD4+ or CD8+ T cells can spare the retina of the injected eye after uniocular anterior chamber inoculation of HSV-1. Because virus appeared after day 7 PI in the ipsilateral optic nerve and retina only in mice depleted of both CD4+ and CD8+ T cells, these results suggest that spread of virus to the ipsilateral retina occurs via the optic nerve and that either CD4+ or CD8+ T cells can prevent spread of virus to the inoculated eye resulting in sparing of the ipsilateral retina.

Dissemination and replication of MCMV after supraciliary inoculation in immunosuppressed BALB/c mice.

PURPOSE: To study replication and dissemination of murine cytomegalovirus (MCMV) in immunosuppressed (IS) and non-IS BALB/c mice after ocular inoculation via the supraciliary route. METHODS: BALB/c mice were immunosuppressed by injections of methylprednisolone, and MCMV was injected via the supraciliary route. Ocular and nonocular tissues from both IS and non-IS mice were studied by plaque assay of tissue homogenates. The frequency of virus-positive leukocytes was determined by PCR. RESULTS: In the inoculated eye, virus replication was significantly higher in both the anterior segment and the posterior segment of IS mice. Virus spread to extraocular sites in both IS and non-IS mice; however, significantly higher titers of virus were recovered from the salivary glands and lungs of IS mice than from non-IS mice, and clearance of virus from these sites was delayed in IS mice. Virus spread from the injected eye via leukocytes, and PCR amplification revealed that the frequency of virus-infected leukocytes was approximately 200-fold higher in IS mice. CONCLUSIONS: The results of these studies suggest that immunosuppression significantly enhances virus replication in the inoculated eye, salivary glands, and lungs, leads to a higher frequency of virus-positive leukocytes, and delays clearance of virus from ocular and nonocular tissues. These results also suggest that retinitis in the injected eye of IS mice correlates with significantly higher titers of virus in the posterior segment.

Modulation of murine herpes simplex virus type 1 retinitis in the uninoculated eye by CD4+ T lymphocytes.

PURPOSE: To investigate the contribution of CD4+ and CD8+ T lymphocytes to retinitis in the contralateral eye after anterior chamber inoculation of herpes simplex virus type 1 (KOS strain). METHODS: T-cell-depleted BALB/c mice were prepared by adult thymectomy and treatment with anti-L3T4 (anti-CD4) monoclonal antibody and/or anti-Lyt2.2 (anti-CD8) monoclonal antibody. On days 9 and 14 after inoculation of herpes simplex virus type 1 (KOS strain) via the anterior chamber, the titer of virus in the uninoculated eye was determined by plaque assay. The eyes were examined microscopically, and the severity of retinitis was evaluated using a histopathologic scoring system. RESULTS: At day 14 postinoculation, significantly less severe destruction of the parenchyma and less inflammatory cell infiltration were observed in the retina of the uninoculated eye of CD4+ T-cell-depleted mice and of mice depleted of both T-cell subsets. The titer of virus in the uninjected eye of CD4+ T-cell-depleted mice and of mice depleted of both CD4+ and CD8+ T cells was also significantly higher at day 14 postinoculation. CONCLUSIONS: The results of the studies suggest three ways CD4+ T cells might contribute to the pathogenesis of herpes simplex virus type 1 retinitis in the uninoculated contralateral eye: (1) accumulation of massive inflammatory cell infiltrates in the retina, (2) induction of retinal destruction, and (3) clearance of herpes simplex virus type 1 from the contralateral eye. In infection of the contralateral retina of nondepleted mice after uniocular anterior chamber inoculation of KOS, CD4+ T cells are required to induce fulminant retinitis and to mediate clearance of virus from the uninoculated eye by day 14 postinoculation. The exact mechanism by which CD4+ T cells contribute to contralateral retinitis remains to be identified.