Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Flap failure after microvascular free-tissue transfer: the fate of a second attempt

In most cases, the loss of a free-tissue transfer is a disaster for both the patient and the surgeon. Seven patients received a second microvascular free-tissue transfer after loss of the first. The indications for free-tissue transfer included chronic osteomyelitis of the lower leg (four patients), acute traumatic defect of the leg (one patient), acute traumatic defect of the arm (one patient), and esophageal defect after surgical excision (one patient). In three patients, the interval between the first and second procedures was less than 2 weeks. The remaining four patients had their second free-tissue transfer performed 5 weeks to 21 months after the first. Six of the seven free flaps were successful. Two patients with venous obstruction occurring after the second free-tissue transfer were salvaged by reexploration. Partial loss of the flap was noted in one of these patients. It is concluded from this select group of patients that failure of a free-tissue transfer does not contraindicate a second microtissue transfer does not contraindicate a second microvascular free-tissue transfer.     

C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase.

C3 nephritic factor (C3NeF) defined by the capacity of nephritic serum and its fractions to initiate loss of the B antigen of C3 in normal serum was purified from the serum of three different donors and shown to function by stabilization of membrane-bound and fluid phase alternative pathway C3 convertase. C3NeF converts cell-bound C3B sites in a dose-related manner to CEB(NeF) sites, which exhibit an approximate 10-fold increase in half-life. The linear relationship between the C3NeF input and the residual hemolytic sites on EAC43B present after incubation for 20 min at 30 degrees C, during which labile C3B sites have decayed, indicates that the number of residual C3B sites is directly related to the dose of C3NeF. The capacity of C3NeF to stabilize the C3B convertase in a temperature- and dose-dependent manner, which is independent of binding or consumption of C3NeF, in a fluid phase reaction mixture of 125I-B, 131I-C3 and D permits isolation of a 10S complex containing radiolabeled C3 and B and exhibiting C3, convertase activity on an exogenous C3 source. Thus, the stabilizing effect of C3NeF is not limited to membrane-bound C3B but is also sufficient to permit recovery of a fluid phase C3 convertase formed during the interaction of C3, B, and D.     

Inhibition of the function of activated properdin by squid chondroitin sulfate E glycosaminoglycan and murine bone marrow-derived mast cell chondroitin sulfate E proteoglycan

We compared the relative capacities of two over-sulfated glycosaminoglycans, heparin and chondroitin sulfate E, to alter the function of native properdin (nP) and activated properdin (aP) in the formation and stabilization of the amplification C3 convertase (C3b,Bb). Heparin was more active on a weight basis than chondroitin sulfate E in inhibiting the formation of C3b,Bb without or with nP, but had no influence on the decay of a pre-formed convertase, either unstabilized or stabilized with nP or aP. In contrast, chondroitin sulfate E was over 10-fold more active than heparin in preventing the formation of C3b,Bb in the presence of aP, and gave dose-related acceleration of decay of pre-formed C3b,Bb,aP but not of unstabilized or nP-stabilized pre-formed convertase. The inhibitory effect of both glycosaminoglycans on the formation of C3b,Bb in the presence of nP or aP was less when the number of C3b sites per target cell was increased. The preferential action of chondroitin sulfate E on the function of aP during the formation and decay of C3b,Bb,aP as compared to C3b,Bb,nP implies functional differences in the two forms of P even when they have been incorporated into C3b,Bb. The equal potency, when adjusted for uronic acid content, of chondroitin sulfate E proteoglycan isolated from the T cell-dependent, bone marrow-derived murine mast cell and of chondroitin sulfate E glycosaminoglycan from squid reveals that the linkage of the glycosaminoglycan to a peptide core does not diminish its regulatory action on the alternative complement pathway.     

Engagement with HIV Prevention Treatment and Care among Female Sex Workers in Zimbabwe: a Respondent Driven Sampling Survey

Objective(S)

To determine the HIV prevalence and extent of engagement with HIV prevention and care among a representative sample of Zimbabwean sex workers working in Victoria Falls, Hwange and Mutare.

Design

Respondent driven sampling (RDS) surveys conducted at each site.

Methods

Sex workers were recruited using respondent driven sampling with each respondent limited to recruiting 2 peers. Participants completed an interviewer-administered questionnaire and provided a finger prick blood sample for HIV antibody testing. Statistical analysis took account of sampling method.

Results

870 women were recruited from the three sites. HIV prevalence was between 50 and 70%. Around half of those confirmed HIV positive were aware of their HIV status and of those 50-70% reported being enrolled in HIV care programmes. Overall only 25-35% of those with laboratory-confirmed HIV were accessing antiretroviral therapy. Among those reporting they were HIV negative, 21-28% reported having an HIV test in the last 6 months. Of those tested HIV negative, most (65-82%) were unaware of their status. Around two-thirds of sex workers reported consistent condom use with their clients. As in other settings, sex workers reported high rates of gender based violence and police harassment.

Conclusions

This survey suggests that prevalence of HIV is high among sex workers in Zimbabwe and that their engagement with prevention, treatment and care is sub-optimal. Intensifying prevention and care interventions for sex workers has the potential to markedly reduce HIV and social risks for sex workers, their clients and the general population in Zimbabwe and elsewhere in the region.