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HIV uses the cell chemokine receptor 5 (CCR5) coreceptor to gain entry to human cells. Researchers studied the infectivity of three strains of HIV in white blood cells and human bone cancer cells. The protease gene of the three subgroups tested were closely related to the MVP-5180 strain of HIV. The tested viral strains did not appear to use cellular fusing, or syncytium formation, to infect the tested cells. All three strains infected peripheral blood mononuclear white blood cells and macrophages.

Chlorpromazine may be capable of preventing HIV from infecting T cells especially when used in conjunction with zidovudine. The initial step in T cell infection occurs when the gp120 viral protein binds to the CD4 T cell receptor. This involves an electrostatic attachment that could be blocked by charged molecules such as chlorpromazine. Researchers studied several derivatives of chlorpromazine and found that 7,8-dioxo-chlorpromazine blocked the attachment of recombinant gp120 and also prevented infection of a T cell culture in the presence of zidovudine.

ABSTRACTTo further refine our current nanoparticle-based HIV-1 p24 antigen assay, we investigated immune responses to p24 to identify diagnostically significant immune dominant epitopes (IDEs) in HIV-infected human sera, to address cross-reactivity of anti-p24 antibodies to different subtypes, and to identify new biomarkers that distinguish acute from chronic HIV infection for more accurate incidence estimation. We identified two major linear epitope regions, located in the CypA binding loop and adjacent helices and at the end of the C-terminal domain. Most sera (86%) from acutely HIV-1-infected individuals reacted with multiple peptides, while 60% and 30% of AIDS patient samples reacted with multiple and single peptides, respectively. In contrast, 46% and 43% of chronically HIV-1-infected individuals reacted with one and none of the peptides, respectively, and only 11% reacted with multiple p24 peptides, indicating a progression of immune responses from polyclone-like during acute infection to monoclone-like or a nonresponse to linear epitopes during chronic infection. Anti-p24 antibodies (subtype B) show broad cross-reactivity to different HIV-1 subtypes, and the synergistic action of different combinations of anti-HIV antibodies improves capture and detection of divergent HIV-1 subtypes. Our results indicate that the modified peptide immunoassay is sensitive and specific for the rapid identification of HIV-1 p24 IDEs and for investigation of immune responses to p24 during natural HIV-1 infection. The data provide the foundation for development and refinement of new assays for improved p24 antigen testing as future tools for rapid and accurate diagnosis as part of early intervention strategies and estimations of incidence.

To determine whether subtypes of HIV-1 and HIV-2 vary in their ability to induce T cell apoptosis in vitro, human peripheral blood mononuclear cells (PBMC) from healthy donors and CEM.NKR-CCR5 cells were infected with a variety of HIV-1 and HIV-2 isolates in vitro. Apoptotic cell levels and chemokine and cytokine production were analyzed. Significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes were observed in PBMCs. The percent of apoptotic cells from each individual ranged from 2 to 78% after HIV-1 infection and from 0 to 28% after HIV-2 infection (p < 0.01). We did not observe significant differences in the degree of apoptosis induced among cells infected with different HIV-1 group M subtypes or group O virus, nor among cells infected with different HIV-2 isolates. However, HIV-2 induced significantly lower degree of apoptosis overall in PBMC and CEM.NKR-CC5 cells when compared with HIV-1 subtypes (p < 0.0001). No significant differences were observed in the production of chemokines, such as RANTES, MIP-1α, and MIP-1β, and cytokines, such as TNF-α and TNF-β when PBMC cultures were infected with different HIV-1 subtype viruses, or HIV-2 isolates. In conclusion, HIV-2 isolates induced significantly lower levels of T cell apoptosis in both PBMC and CEM.NKR-CCR5 cells than HIV-1 isolates. No differences in T cell apoptosis levels were seen between different subtypes of HIV-1 group M or group O isolates. This is consistent with the mild clinical course of infection with HIV-2 that has been reported relative to that observed with HIV-1.

We used patch clamp electrophysiology and concurrent imaging with the Ca-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca concentration ([Ca] i ) during mouse egg fertilization. We found an ∼2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca] i at sperm fusion. This was followed ∼1 min later by a rise in [Ca] i that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-μM peak [Ca] i favored capacitance decrease, while higher [Ca] i favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca] i . The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca] i dependence. Raising [Ca] i by the intracellular introduction of IP 3 initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca] i was increased modestly by activation of an endogenous Ca current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca] i and that sperm and the active component of sperm factor may be doing more than initiating the IP 3 -mediated release of intracellular Ca.

We used patch clamp electrophysiology and concurrent imaging with the Ca2+-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca2+concentration ([Ca2+]i) during mouse egg fertilization. We found an ∼2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca2+]iat sperm fusion. This was followed ∼1 min later by a rise in [Ca2+]ithat led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-μM peak [Ca2+]ifavored capacitance decrease, while higher [Ca2+]ifavored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca2+]i. The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca2+]idependence. Raising [Ca2+]iby the intracellular introduction of IP3initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca2+]iwas increased modestly by activation of an endogenous Ca2+current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca2+]iand that sperm and the active component of sperm factor may be doing more than initiating the IP3-mediated release of intracellular Ca2+.

Extracellular ATP activates large increases in cell surface area and membrane turnover in rat brown adipocytes (Pappone, P. A., and Lee, S. C. 1996. J. Gen. Physiol. 108:393–404). We used whole-cell patch clamp membrane capacitance measurements of membrane surface area concurrently with fura-2 ratio imaging of intracellular calcium to test whether these purinergic membrane responses are triggered by cytosolic calcium increases or G protein activation. Increasing cytosolic calcium with adrenergic stimulation, calcium ionophore, or calcium-containing pipette solutions did not cause exocytosis. Extracellular ATP increased membrane capacitance in the absence of extracellular calcium with internal calcium strongly buffered to near resting levels. Purinergic stimulation still activated exocytosis and endocytosis in the complete absence of intracellular and extracellular free calcium, but endocytosis predominated. Modulators of G protein function neither triggered nor inhibited the initial ATP-elicited capacitance changes, but GTPγS or cytosolic nucleotide depletion did reduce the cells’ capacity to mount multiple purinergic responses. These results suggest that calcium modulates purinergically-stimulated membrane trafficking in brown adipocytes, but that ATP responses are initiated by some other signal that remains to be identified.

Abstract:  We used whole-cell and perforated-patch voltage-clamp methods to study the membrane electrical properties of isolated rat epididymal and inguinal white adipocytes. We examined cells from both Sprague-Dawley and Zucker lean and Zucker obese (fa/fa) rats. A delayed-rectifier potassium current was present and similar in unstimulated white fat cells from all these sources. The potassium current activated rapidly with depolarization positive to about –30 mV and showed slow inactivation. Stimulation with extracellular ATP activated both hyperpolarizing and depolarizing conductances. ATP exposure also increased cell membrane capacitance by an average of 16%, suggesting that ATP activates exocytosis. Exposure to norepinephrine had little electrophysiological effect. We conclude that white adipocytes are very similar to brown adipocytes in their resting electrophysiological profile and in their responses to extracellular ATP.

Forskolin, a direct activator of adenylate cyclase, modifies the voltage-dependent K+ conductance of quiescent human peripheral blood T lymphocytes. In the presence of greater than 20 µM forskolin, the average voltage-gated current in whole-cell patch clamp is significantly decreased. The voltage dependence and kinetics of activation are not changed from untreated control cells. However, inactivation becomes biphasic. Much of the current inactivates very quickly (complete in 10 ms), and the remaining outward current inactivates more slowly with a time constant closer to that of control cells. To determine whether this effect is mediated by a rise in intracellular cAMP, cells were preincubated and subsequently voltage-clamped in the presence of other agents that raise the cAMP levels in T cells (isoproterenol plus a phosphodiesterase inhibitor, or dibutyryl cAMP) with no effect on the K+ conductance. Similarly, cells put in whole-cell patch clamp with cAMP, GTP, ATP, and theophylline added to the electrode filling solution showed no change in K+ current. Because other proccdures that raise cAMP did not duplicate the effect of forskolin, we investigated the effect of 1,9-dideoxyforskolin, an analogue of forskolin that does not stimulate adenylate cyclase in human lymphocytes. This drug induced changes in the whole-cell K+ conductance identical to those observed with forskolin. Both forskolin and dideoxyforskolin inhibit mitogen-induced proliferation of lymphocytes. Because inhibition of proliferation occurs in the presence of known K+ channel blockers, these results suggest that forskolin has an effect on T cell mitogenesis that is mediated by inhibition of K+ conductance and is independent of cAMP.

Several diagnostic assays for the detection of HIV infection have been approved and licensed by the FDA for blood donor screening. However, the performance of these assays is unknown when testing genetically divergent blood specimens. To evaluate the performance of these assays with diverse HIV strains, we chose to study specimens collected from blood donors in Cameroon where genetic diversity and recombinant variants are prevalent. In this study, we tested 240 human plasma specimens collected from two blood centers in Cameroon. These samples were screened initially in Cameroon for antibody to HIV using a rapid assay. We also performed sequencing to determine subtype. Our evaluation has demonstrated that HIV infection in most HIV plasma samples could be detected by most of the US FDA licensed diagnostic assays. With the exception of a few specimens, HIV‐1 p24 antigen was not detected in any of the samples. In addition, some nucleic acid tests (NAT) assays were not able to detect a few serologic reactive samples and all new variants including some CRF02_AG variants. J. Med. Virol. 78:S22–S23, 2006. © 2006 Wiley‐Liss, Inc.

Although there is no evidence for transmission of mammalian retroviruses to humans via vaccine immunization, the allegations of contamination of oral poliovirus vaccines with human immunodeficiency virus (HIV) type 1 or a hypothetical progenitor virus from monkeys has created controversy and dispute regarding the origin of AIDS in humans. Twelve monovalent lots of live, attenuated oral poliovirus vaccine types 1, 2, and 3, which were released for use by a North American manufacturer between 1976-1989, were tested for the presence of HIV-1 and simian immunodeficiency virus (SIV). HIVISIV were not detected in these monovalent poliovirus vaccine lots with the reversetranscriptase assay, a general detection assay, and highly sensitive and specific polymerase chain reaction assays.