New insights into the immunology and evolution of HIV

Fewer than one million HIV infected individuals are currently receiving anti-retroviral therapy.The limitations of such treatment have underscored the need to develop more effective strategies to control the spread and pathogenesis of HIV.Typically,naturally occurring protective immune responses provide the paradigm for such development.It is now clear however that HIV can utilise the millieu of and activated immune system to its own replicative advantage.Mobilisation of the immune response,intended to thwart the virus,may instead fuel its dissemination. ‘immune escape’and spread.The immense genetic variation of HIV contributes to lack of immune control and the development of progressive disease in the majority of infected,untreated individuals.Further delineation of the intimate interactions between the HIV and the immune system will be critical and recent advances in this direction are discussed.     

Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.     

Protein dynamics in a family of laboratory evolved thermophilic enzymes

Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 degrees C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm(-1)), with the more stable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Mining the Giardia lamblia genome for new cyst wall proteins

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by approximately 50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

Differential degradation of amyloid beta genetic variants associated with hereditary dementia or stroke by insulin-degrading enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.     

Assessment of thin-layer breast aspirates for immunocytochemical evaluation of HER2 status

OBJECTIVE: To determine whether immunocytochemistry (ICC) for HER2 on ThinPrep (TP)-processed breast fine needle aspiration biopsies (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) is comparable to the findings of immunohistochemistry on corresponding surgically removed tissue. STUDY DESIGN: Immunostaining was performed on 63 malignant breast fine needle aspirates and compared to immunostaining on paraffin sections (PSs) from the subsequent biopsies. The HercepTest (Dako, Carpinteria, California, U.S.A.) and TAB250 antibodies were utilized. Cases in which the TP and paraffin HER2 results did not correlate were further assessed for gene amplification by differential polymerase chain reaction (dPCR). RESULTS: HER2 overexpression was found in 9 of the 63 cases (14%). TAB250 had higher specificity on PS versus TP (P = .008), and TAB250 had higher specificity on PS versus the HercepTest on PS and TP (P = .004 and .0001, respectively). CONCLUSION: HER2 immunostaining with both the HercepTest and TAB250 on TP is unreliable due to low specificity (72% and 83% for HercepTest and TAB250, respectively). However, both antibodies have high sensitivity (89% and 100%, respectively); suggesting that this method may have some utility as a preliminary screening test for HER2 status. Negative HER2 staining by ICC is highly predictive of the absence of HER2 overexpression, whereas positive HER2 staining on TP would require further validation by either dPCR of fluorescence in situ hybridization.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.