We studied the evolutionary relationships between the two protease inhibitor (PI) resistance mutations, D30N and L90M, of human immunodeficiency virus type 1 (HIV-1). The former is highly specific for nelfinavir resistance, while the latter is associated with resistance to several PIs, including nelfinavir. Among patients with nelfinavir treatment failure, we found that D30N acquisition was strongly suppressed when L90M preexisted. Thus, D30N/L90M double mutations not only were detected in a very limited number of patients but also accounted for a minor fraction within each patient. In the disease course, the D30N and L90M clones readily evolved independently of each other, and later the D30N/L90M double mutants emerged. The double mutants appeared to originate from the D30N lineage but not from the L90M lineage, or were strongly associated with the former. However, their evolutionary pathways appeared to be highly complex and to still have something in common, as they always contained several additional polymorphisms, including L63P and N88D, as common signatures. These results suggest that D30N and L90M are mutually exclusive during the evolutionary process. Supporting this notion, the D30N/L90M mutation was also quite rare in a large clinical database. Recombinant viruses with the relevant mutations were generated and compared for the ability to process p55 gag and p160 pol precursor proteins as well as for their infectivity. L90M caused little impairment of the cleavage activities, but D30N was detrimental, although significant residual activity was observed. In contrast, D30N/L90M demonstrated severe impairment. Thus, the concept of mutual antagonism of the two mutations was substantiated biochemically and functionally.

Thrombi, encapsulated hematomas, and granulation tissue are frequently seen in cerebral cavernous malformations (CCMs). We investigated the role that these histological changes play in repeated hemorrhages in CCMs as well as lesion growth, examining specimens of CCMs surgically harvested from 20 patients. The immunohistochemical study included thrombomodulin (TM) and endothelial cell protein C receptor (EPCR), which are important regulators of blood coagulation. Thick capsules, which contained blood degradation product, were seen in cases with encapsulated hematomas. Clusters of sinusoidal vessels were found outside of these thick capsules. Granulation tissue with inflammatory infiltrates and capillaries was seen in 4 cases with non-capsulated hematomas. Organizing thrombi were seen in sinusoidal vessels in 15 out of 20 cases. Factor VIII-related antigen staining demonstrated numerous capillaries in and around organizing thrombi and within the thickened vessel walls as well as in both the inner and outer sides of the hematoma capsule. TM and EPCR were positive in the endothelial cells of these capillaries, whereas they were negative in those of capillaries in the brain surrounding the lesions. Our study suggests that thrombosed sinusoidal blood vessels could gradually expand by repeated bleeding from numerous capillaries inside the wall and become encapsulated hematomas, and capillaries outside the thickened vessel wall could become sinusoidal blood vessels. Thrombosis within cerebral venules could be one of the causal factors of CCMs.

Fat infiltration within muscle could lead to inhibition of normal activity of musculature. The present study suggests that fat within cervical multifidus muscle could directly cause postural instability in static standing, even though the proprioceptive information has normal lower limbs.

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