Positively charged amino acid substitutions at positions 11 and 25 within the loop of the third variable region (V3) of HIV-1 subtype B envelope have been shown to be associated with the syncytium-inducing (SI) phenotype of the virus. The present study was designed to examine SI and NSI-associated V3 mutations in HIV-1 subtypes other than B. HIV-1 RNA was isolated from 53 virus stocks and 26 homologous plasma samples from 53 recently infected individuals from Brazil, Rwanda, Thailand, and Uganda. The C2-V3 region of the viral envelope was converted to cDNA, amplified, and sequenced. Of 53 primary virus stock samples 49 were biologically phenotyped through measurement of the syncytium-inducing capacity in MT-2 cells (to differentiate between SI and NSI phenotypes). In addition, after passage of primary isolates through PHA stimulated donor PBMC, the replication capacity was determined in U937-2, CEM, MT-2, and Jurkat-tat cell lines (to differentiate rapid/high and slow/low phenotypes). According to the sequence analysis 9 (17.0%) of the viruses belonged to subtype A, 15 (28.3%) to subtype B, 1 (1.9%) to subtype C, 13 (24.5%) to subtype D, and 15 (28.3%) to subtype E. Sequence analysis of virus RNA, obtained from 26 homologous plasma samples, confirmed the homogeneity of sequence populations in plasma compared to primary virus isolates. Of the 49 viruses tested 12 had the SI phenotype, 5 were confirmed to be rapid/high, and 4 appeared to be slow/low pattern 3 replicating. Of 49, 29 had the NSI phenotype, 24 were confirmed to be slow/low pattern 1 or 2, and 3 appeared to be slow/low pattern 3 replicating. Analysis of mutations at V3 loop amino acid positions 11 and 25 revealed that 10/12 (83.3%) of the SI viruses had SI-associated V3 mutations and that 28/29 (96.6%) of the NSI viruses lacked these mutations. V3 loop heterogeneity, length polymorphism, and a high number of positively charged amino acid substitutions were most frequently found among subtype D variants. These results indicate that both the phenotypic distinction between SI and NSI viruses and the association of biological phenotype with V3 mutations is present among HIV-1 subtypes other than B.

Arguments for an involvement of bacteria in the pathogenesis ofchronicjoint inflammation (arthritis) are several (1-12). To study the pathogenetic mechanism ofbacteriuminduced sterile arthritis, different animal models are used, among which, Mycobacterium tuberculosis-induced or adjuvant arthritis (AA)t (2) and streptococcal cell wall (SCW)-induced arthritis (1) are the best known. In both models, bacterium-specific T lymphocytes have been demonstrated to play a crucial role (13-24, and van den Broek, M. F, M. C. J. van Bruggen, A. J. Severijnen, and W B. van den Berg, manuscript submitted for publication), and one aspect ofthis role might be the crossreactive nature of these T lymphocytes to cartilage-associated components (22, 25).In AA it is a well known fact that rats that have recovered from arthritis are resistant to a subsequent induction of AA (17,18), and it has been demonstrated recently that pretreatment of rats with the mycobacterial 65-kD protein resulted in the same resistance to AA when tested 35 d later (13) . Since the 65-kD protein is an ubiquitous bacterial common antigen (26), with a homologous molecule present also in streptococci, and probably even associated with cell walls (27), we tested the effects ofpretreatment with the mycobacteria165-kD protein on the development of SCW arthritis.Here we show that intraperitoneal administration of 50 lAg 65-kD protein to rats 35, 25, 15, or even as short as 5 d before induction of SCW arthritis completely protected the rats against the chronic erosive polyarthritis. The resistance against SCW arthritis is transferrable by spleen T cells to naive recipients . The protection coincides with a suppression of SCW-specific T cell responses, thus displaying features much alike suppression or tolerance leading to resistance in other models for autoimmune diseases (28-31).

To gain insight into determinants that define the duration of the asymptomatic period preceding AIDS, groups of long-term asymptomatic (LTA) person (> 7 years of follow-up) and slow and rapid progressors of human immunodeficiency virus infection were studied. LTAs had no clinical manifestations of AIDS or immunologic abnormalities in 7 years of follow-up. RNA copy numbers, gag- and env-specific, and neutralizing antibody titers in serum were determined 1 and 5 years after seroconversion or entry into the cohort. Early in infection, before immunologic markers or clinical manifestations allowed group discrimination, subjects who were later classified as LTAs had significantly less serum viral RNA than progressors. No significant increase in virus load was found in progressors, indicating that the initial load defines clinical outcome. In slow progressors, high virus load was associated with high p24-specific antibody titers, suggesting that delay of clinical manifestations of AIDS may be related to the presence of high levels of p24-specific but not V3-specific antibodies.

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Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.

The prevalence of HIV-1 sequences of the envelope domains V1V2 and V3 was analyzed by RT-PCR amplification. Two distinct biological phenotypes of HIV-1 have been described: the nonsyncytium-inducing (NSI) phenotype, best characterized by the inability to infect MT-2 cells, and the syncytium-inducing phenotype (SI), with the ability to infect MT-2 cells. Viral phenotype SI has been associated with HIV pathogenesis. The presence of positively charged amino acids at position 306 and 320 in the V3 domain of gp120 has been shown to be required for the support of the SI phenotype. In addition, V2 elongation and relocation of N-glycosylation sites were postulated to herald an NSI to SI phenotype switch. The present study was designed to assess the stability of an elongated V2 region with relocated N-glycosylation sites observed in SI isolates compared to NSI isolates. Eleven isolates with the SI phenotype and 19 isolates with the NSI phenotype were included in the study. Nine of the SI and 1 of the NSI isolates had a positively charged residue at position 306 or 320 (p < 0.001) in the V3 domain as assessed by direct sequencing of the viral RNA. In contrast, elongation and/or relocation of N-glycosylation sites of the V2 variable region were not found to be a consistent genetic feature of the SI phenotype. However, SI isolates had more positively charged amino acid residues in the hypervariable V2 region compared with NSI isolates. In one of the two SI isolates lacking positively charged amino acids at positions 306 or 320 in the V3 loop an elongation of 26 amino acids with 4 additional N-linked glycosylation sites was observed in the V2 region. This is consistent with the theory that elongation of V2 may be transiently required for SI conversion. These results suggest that maintenance of the SI phenotype requires positively charged amino acids in V3 in the majority of the virus population, but not an elongated V2 region with added or relocated N-linked glycosylation sites. Although increased charged residues in the V2 region may contribute.

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The mycobacterial 65 kDa heat shock protein (HSP65) is of critical significance in the model of adjuvant arthritis (AA). Arthritogenic and protective T cell clones obtained from arthritic rats recognized the 180-188 sequence of HSP65. Previous reports have shown that administration of HSP65 prior to disease induction led to resistance to arthritis in the AA model and in several other models of experimental arthritis. Here, we report the development of immunity to HSP65 and the critical 180-188 epitope during the course of AA. Following Mycobacterium tuberculosis (MT) immunization both antibodies and T cell responses to HSP65 were detected. Proliferative responses to the 180-188 epitope were seen exclusively in the local draining lymph node cells at day 14 after immunization. The anatomical distribution and course of T cell responses to HSP65 and its 180-188 epitope are compatible with T cell regulated control of the disease. Although lower HSP65 antibody levels were observed in the animals with severe arthritis, in individual animals no evidence was obtained for a relationship between development of HSP65 humoral immunity and arthritis severity. Nevertheless, during disease exacerbation, elicited by HSP65 immunization during disease development, elevated T cell responses against HSP65 and its 180-188 epitope were found. In contrast, we obtained evidence that successful transfer of arthritis resistance to naive recipients depends on the transfer of HSP65 specific T cells. On the basis of these results, it seems that HSP65 plays a crucial role in the T cell regulatory events involved in both the induction of, and protection against, AA.(ABSTRACT TRUNCATED AT 250 WORDS)