Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and phosphorylation.

Effects of intestinal ischemia-reperfusion injury on rat peripheral blood neutrophil activation.

Peripheral blood neutrophil activation status is indicative of remote organ damage after intestinal ischemia secondary to aortic aneurysm repair. However, the effects of direct intestinal ischemia-reperfusion (I/R) injury on neutrophil activation and its reflection of remote organ injury have not been evaluated. DA rats were subjected to 30 min of intestinal ischemia or sham surgery. Blood samples were taken before ischemia and 30, 60, and 120 min after reperfusion. Neutrophil counts were quantified and CD11b, CD62L, and NKR-P1 expression was assessed using flow cytometry. The sham procedure induced increases in neutrophil numbers (P < 0.001), which was transiently attenuated in animals subjected to intestinal I/R injury. CD11b expression increased in both groups, whereas CD62L and NKR-P1 (P < 0.01) expression decreased in both groups. These findings suggest that even mild surgical procedures induce demargination of neutrophils. Monitoring the peripheral blood for activated neutrophils is of no value in assessing the severity of direct intestinal I/R injury or predicting remote organ damage after intestinal ischemia.

Neonatal disease induced by SIV infection of the rhesus monkey (Macaca mulatta).

Seven 72-hr-old Indian origin rhesus monkeys (Macaca mulatta) were inoculated with 10 animal ID50 of SIV/DeltaB670. Nine age-matched animals were used as uninoculated controls. All seven inoculated animals became infected as verified by viral isolation and SIV p26 antigenemia. Five of seven infected animals died within a mean of 31 days (range, 26-41 days), with high levels of antigenemia beginning 1-2 weeks postinoculation (PI) that persisted until death. Absolute lymphocyte numbers were within normal limits in all animals in both groups throughout the study. Inoculated animals that died within a mean of 31 days (short-term survivors) had significantly lower numbers of CD4+CD29+ (helper/inducer) lymphocytes than did long-term surviving inoculated animals through 3 weeks PI. Numbers of CD4+ lymphocytes were no different when controls were compared to all inoculated animals through 4-5 weeks PI. The two inoculated animals surviving 216 and 423 days PI (long-term survivors) did demonstrate declining CD4+ cells, but only late in disease. CD8+ lymphocytes were significantly lower in short-term survivors when compared to long-term survivors through 5 weeks PI. Antibody production against SIV viral proteins was detected only in the long-term survivors and was similar to results from past studies in juveniles. Clinical signs in the inoculated group were consistent with those seen in past studies on older animals. Persistent bacterial infections, primarily of the GI and respiratory tracts, were seen in the infected group. Aside from the lack of some opportunistic infections such as cytomegalovirus (CMV) and Pneumocystis carinii, necropsy findings were not different when compared to past studies on juvenile animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of initiation of 3'-azido,3'-deoxythymidine (zidovudine) treatment at different times after infection of rhesus monkeys with simian immunodeficiency virus.

The effects of initiating treatment with 3'-azido-3'-deoxythymidine (zidovudine) at different times after inoculation of simian immunodeficiency virus (SIV) were investigated in rhesus monkeys. Zidovudine treatments of 100 mg/kg/day (25 mg/kg, subcutaneously every 6 h) were initiated 1, 8, 24, or 72 h after intravenous inoculation of 10 ID50 of SIV. Treatments continued for 28 days, and results were compared with those of saline-treated controls. Serum infectious virus titers 14 days after inoculation (AI) significantly decreased after treatment initiation 1, 8, or 24 h AI. Titers were correlated with the time treatment was initiated. Treatments initiated 1-72 h AI prevented the establishment of persistent SIV antigenemia; greater effects were observed with earlier initiation of treatment. Treatments initiated 1-8 h AI resulted in decreased levels of viral antigenemia 14 days AI and delayed decreases in CD4+CD29+ blood lymphocytes. Earlier treatment initiation resulted in delayed recurrence of antigenemia, with a tendency for longer survival. Early initiation of treatment may be important for limiting initial viral replication and dissemination in cases of known exposure.

Thymus in simian immunodeficiency virus-infected rhesus monkeys.

The thymuses from 20 simian immunodeficiency virus (SIV)-infected and 4 uninfected rhesus monkeys were examined at intervals after infection to determine whether there were specific SIV-induced lesions, to document the serial distribution of SIV antigens, mRNA, and DNA, to quantitate the number of infected cells, and to correlate thymic changes with other parameters of infection. The following techniques were used: gross pathology, histopathology, immunohistochemistry, electron microscopy, in situ hybridization, polymerase chain reaction, and limiting dilution culture. Thymic involution due to loss of lymphocytes was apparent 8 weeks after inoculation. No epithelial damage or loss of Hassall's corpuscles was observed. Culture was the most sensitive technique for detecting SIV, being positive in 19 of 20 inoculated monkeys. The polymerase chain reaction was negative in one thymus that was positive at a low level by culture. In situ hybridization was positive in 14 of 19 thymuses examined, with a few macrophages in the cortex having a strong signal and numerous lymphocytes in the medulla having a weak signal. Mature viral particles and viral budding could not be demonstrated by electron microscopy. The number of cells positive for viral RNA by in situ hybridization correlated with the level of serum antigenemia. These observations suggest that thymic macrophages and lymphocytes are infected with SIV within 2 weeks after inoculation. SIV apparently directly causes loss of thymic lymphocytes and immunodeficiency without infecting or damaging the thymic epithelium. No specific SIV-induced lesions were recognized. The number of cells in the thymic medulla expressing SIV RNA correlates with the level of serum antigen, which has been previously shown to be correlated with disease progression.

Infection of rhesus monkeys with topical instillation of simian immunodeficiency virus (SIV)B670 into the conjunctival sac.

We tested the ability of SIV to cause local and systemic infection in three rhesus monkeys after topical instillation of cell-free virus into the conjunctival cul-de-sac. Conjunctivitis or other signs of infection were monitored after inoculation. Conjunctiva were swabbed for virus culture and biopsied for PCR. Changes in lymphocyte subsets, seroconversion, antigenemia, and virus isolation from PBL were assessed systemically postinoculation. Viral DNA was detected in conjunctival biopsy by PCR in one of three animals that later developed systemic infection. The other two animals remained uninfected. These data demonstrate that the conjunctiva is a route by which SIV (and perhaps HIV) may cause systemic infection.

Efficacy of SIV/deltaB670 glycoprotein-enriched and glycoprotein-depleted subunit vaccines in protecting against infection and disease in rhesus monkeys.

Immunization with an inactivated whole-virus vaccine is highly effective in preventing lentivirus infection. The viral protein(s) essential to the induction of protective responses, however, have not been identified. To define the role of virion components in the induction of protective immunity, we evaluated the efficacy of glycoprotein-enriched and glycoprotein-depleted simian immunodeficiency virus (SIV) subunit vaccines prepared by lentil-lectin affinity chromatography of gradient-purified virions using the immunization and challenge regimen previously found successful with an inactivated whole-virus vaccine. Infection was determined by successful recovery of virus, the induction of SIV-specific antibody responses, and infection of naive recipients by inoculation with lymph-node-derived lymphocytes from the vaccinates. Immunization with the glycoprotein-enriched preparation prevented infection in two out of four monkeys, whereas the glycoprotein-depleted vaccine failed to prevent infection in all four vaccinates tested. However, the glycoprotein-depleted vaccine appeared to moderate the progression of SIV-induced disease compared with non-immunized infected control monkeys inoculated with the same challenge dose. These data suggest that subunit vaccines containing sufficient quantities of viral glycoproteins can protect against SIV infection, whereas subunit vaccines composed predominantly of viral core proteins cannot. The development of effective vaccines against HIV infection should include studies on the optimum presentation of the viral envelope glycoproteins to produce long-term broadly protective immune responses.

Ocular manifestation of simian immunodeficiency syndrome (SAIDS).

The management of opportunistic infections is a significant problem in acquired immunodeficiency syndrome (AIDS) and the development of more effective chemotherapeutic agents is needed. We present the ocular manifestations of an AIDS-like disease in rhesus monkeys experimentally infected with simian immunodeficiency virus (SIV) at the Delta Regional Primate Research Center. These findings consisted of rubeosis in the anterior segment and retinitis, optic neuritis, choroiditis and panophthalmitis in the posterior segment of the eye. Investigation of the retinas by electron microscopy revealed SIV in both eyes of one animal and a herpes virus in two animals. Serology confirmed cytomegalovirus (CMV) as the likely agent. This primate model will prove useful for both further investigations of the possible interaction between immunosuppressive lentiviruses and CMV in ocular disease and antiviral drug testing.

Experimental infection of timed-pregnant rhesus monkeys with simian immunodeficiency virus (SIV) during early, middle, and late gestation.

Ten rhesus monkeys were inoculated with SIV/DeltaB670 during various stages of gestation to determine factors predictive of transplacental infection. Two abortions associated with rapid disease occurred shortly after infection; uninfected infants were caesarean delivered from eight other females. SIV-specific RNA accompanied by deciduitis was identified in the maternal portion of two placentas suggesting that opportunistic infections may promote entry of SIV into placental tissue. The lack of evidence for SIV infection of caesarean delivered infants suggests that fetal infection may often occur during parturition.

A formalin-inactivated whole SIV vaccine confers protection in macaques.

A vaccine against human immunodeficiency virus (HIV) would be highly effective in stopping the acquired immunodeficiency syndrome (AIDS) epidemic. A comprehensive evaluation of potential vaccine methodologies can be made by means of the simian model for AIDS, which takes advantage of the similarities in viral composition and disease potential between simian immunodeficiency virus (SIV) infection of rhesus macaques and HIV infection in humans. Immunization with a formalin-inactivated whole SIV vaccine potentiated with either alum and the Syntex adjuvant threonyl muramyl dipeptide (MDP) or MDP alone resulted in the protection of eight of nine rhesus monkeys challenged with ten animal-infectious doses of pathogenic virus. These results demonstrate that a whole virus vaccine is highly effective in inducing immune responses that can protect against lentivirus infection and AIDS-like disease.

Spinal cord compression by a rheumatoid nodule.

A case, believed to be unique, is reported of spinal cord compression due to an extradural rheumatoid nodule.

Malignant gliomas occurring in identical twins.

The occurrence of malignant gliomas in a pair of identical twins is reported. One, in the posterior fossa, presented in the third year of life, the other, in the left cerebral hemisphere, in the eighth. Both ran a rapidly progressive course and died within a few months of the onset of symptoms. The histological structure of the gliomas was similar and both tumours consisted of astrocytic and oligodendroglial parts. Review of the literature revealed only two pairs of histologically proven gliomas occurring in identical twins. The number of cases reported is too small for any valid conclusions on possible genetic mechanisms.