Preoperative gentling does not attenuate septal-lesion induced hyperreactivity

Rats were either gentled or not gentled for 10 minutes a day for 7 days. At the end of this time a lesion was made in the region ventral to the anterior septum or in the lateral septum, or the rat was given a sham lesion. Reactivity to the experimenter was tested at 2, 7, and 14 days postoperatively. The reactivity scores of animals gentled preoperatively were not different from those of animals which had not been gentled but the animals of both groups were significantly more reactive than animals given a sham lesion. It is concluded that preoperative handling does not attenuate the increase in defensive behavior induced by lesions of the lateral septum or of the region ventral to the anterior septum.     

Mouse killing and hyperreactivity following lesions of the medial hypothalamus, the lateral septum, the bed nucleus of the stria terminalis, or the region ventral to the anterior septum

Electrolytic lesions in the medial hypothalamus, the lateral septum, or the region ventral to the anterior septum induced home cage mouse killing in 85 to 100% of rats when tested 2 days postoperatively and in 50 to 70% when tested at 21 days. When tested in a novel and larger test chamber, 100% of the rats with medial hypothalamic lesions killed mice at 2 days postoperatively but only 30% killed at 21 days postoperatively. Ten to thirty percent of animals with lesions of the lateral septum or of the region ventral to the anterior septum killed at any time in the novel environment. Lesions of the stria terminalis produced a slight increase in mouse killing in both the home cage and the novel environment. A high level of reactivity was produced by lesions of the medial hypothalamus, the lateral septum or the region ventral to the anterior septum but only that caused by the medial hypothalamic lesions was sustained over the 21 day test period. These results support previous evidence that the lateral septum, the region ventral to the anterior septum, and the medial hypothalamus are each important areas modulating mouse killing.     

Virus replication and evolution drive the kinetics and specificity of SIV-specific cytotoxic T lymphocytes

Summary: SIV (simian immunodeficiency virus) infection of cynomolgus macaques provides an excellent model for investigating the basis of protective immunity against HIV (human immunodeficiency virus). We explored the protective role of cytotoxic T lymphocytes (CTL) against the pathogenic molecular clone SIVmac-J5. Vaccine-induced CTL precursors (CTLp) against Env, Gag or Nef did not protect macaques against intravenous challenge. However, detection of Rev-specific CTLp in infected macaques was associated with effective virus containment. Furthermore, CTL against an immunodominant Gag/p26 epitope (amino acids 242–250) resulted in the emergence of a mutant virus that uniformly replaced wild-type virus in the spleen and partially escaped recognition. During primary infection, CTLp detection in blood coincided with decreasing viremia. After 12 months, two outcomes emerged. In one group of macaques, persistent viremia was associated with high viral load in lymphoid organs and declining CD4⁺ T-cell counts. CTLp were maintained in asymptomatic macaques, but declined in the symptomatic phase of infection. In a second group, loss of detectable viremia was associated with low-level virus reservoirs in lymphoid organs, asymptomatic status and maintained CD4⁺ T-cell counts. CTLp peaked in the first 4 months of infection and subsequently declined in this group. These studies provide insights into the complex interplay between virus replication and host immunity.     

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.     

Tranexamic acid: Preoperative prophylactic therapy for patients with hereditary angioneurotic edema

Short-term therapy of 96-hr duration with tranexamic acid was prophylactically effective as defined by the absence of attacks of angioedema in 14 patients with hereditary angioedema undergoing 10 dental and 4 general surgical procedures. Eight of the 14 patients had previously undergone dental extractions without prophylactic therapy with antifibrinolytic agents and each had experienced one or more attacks of angioedema. Seven of these 8 patients had a cumulative experience of 13 episodes of laryngeal edema after dental extractions and the eighth had a bout of cutaneous angioedema. Although the number of dental extractions conducted without prophylactic antifibrinolytic therapy cannot be accurately defined in retrospect, the prominence of laryngeal edema in this circumstance is striking when compared with the absence of attacks in the presence of prophylaxis with tranexamic acid. Methyltestosterone and impeded androgens are now known to be effective prophylaxis for spontaneous and, presumably, postoperative attacks when employed chronically because their administration is associated with correction of the biochemical defect of hereditary angioneurotic edema, but their chronic administration to children and women of childbearing age requires further definition because of their potential pituitary suppressive action. Tranexamic acid prophylaxis makes it possible to offer to untreated patients with hereditary angioneurotic edema dental work and other operative procedures that in the past were withheld or conducted with considerable risk.     

Truncated immunoglobulin Dmu causes incomplete developmental progression of RAG-deficient pro-B cells.

Early stages of B cell development are dependent on the expression of a pre-B cell receptor (BCR), composed of a mu heavy chain (HC) in association with surrogate light chain (SLC) proteins and the signaling molecules, Igalpha and Igbeta. During the formation of the variable region of the mu chain by somatic gene rearrangement, a truncated form of the mu protein (called Dmu) is sometimes produced by the rearrangement of a D(H) segment to a J(H) segment using one of three reading frames (designated rf2). When a Dmu protein is formed, subsequent B cell development is blocked by down-regulation of further HC rearrangements, so that a full-length muHC cannot be formed. In this study, we demonstrate that in recombinase activating gene (RAG)-2-deficient B220(+) CD43(+) pro-B cells in which B lymphopoiesis has been arrested at fraction C, transgenic expression of Dmu promoted partial developmental progression to fraction C', but was unable to mediate the pro-B to pre-B cell transition to fraction D effected by full-length muHC protein. These data suggest that the intracellular signaling pathways engaged by the Dmu pre-BCR are insufficient to facilitate the expansion and/or survival of pre-B cells, and are distinct from those engaged by the pre-BCR-containing full-length muHC.     

Immunohistochemical and molecular analysis of p53, RB,and PTEN in malignant peripheral nerve sheath tumors

The molecular basis of both sporadic and neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) is yet largely undetermined. Therefore, we analyzed a series of 12 MPNSTs - including two cases which arose in the setting of NF1 - for molecular alterations in the p53, retinoblastoma ( Rb), and PTEN tumor suppressor genes. Furthermore, the immunohistochemical expression of p53, RB, and PTEN protein was examined in these tumors. One mutation (8%), an A to T transversion leading to an amino acid exchange, was found in exon 5 of the p53 gene in a sporadic MPNST. In two other sporadic tumors (20%), loss of heterozygosity (LOH) of the p53 gene occurred. Nuclear overexpression of p53 protein was observed in ten tumors (83%). Loss of RB protein expression was seen in two MPNSTs (17%), and LOH of the Rb gene was detected in four tumors (44%), including the two NF1-associated MPNSTs, one of them showing concomitant loss of RB protein expression. No mutation in the PTEN gene was detected, and cytoplasmic immunoreactivity for the PTEN protein was maintained in eight MPNSTs (67%). We suggest that alterations in the p53 and RB pathway, both are essential in controlling the cell-cycle progression, are critical points in the tumorigenesis of sporadic and NF1-associated MPNSTs, whereas the PTEN gene seems to play no significant role in this process.     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.