The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity.

We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo.Introduction: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. Materials and Methods: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n ‫ס‬ 80) or 7 mo later (restoration mode; n ‫ס‬ 152). Hind limbs and lumbar spine BMD was measured at 2-or 4-wk intervals in vivo and ex vivo on termination at 8-16 wk. Long bones were subjected to CT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. Results: In the prevention mode, low doses (0.1 and 0.3 g) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 g TSH had increased BMD (10-11%), trabecular bone volume (100-130%), trabecular number (25-40%), trabecular thickness (45-60%), cortical thickness (5-16%), mineral apposition and bone formation rate (200-300%), and enhanced mechanical strength of the femur (51-60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. Conclusions: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling.

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro 3 Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.osteoclast ͉ osteoporosis ͉ pituitary ͉ osteoblast ͉ bisphosphonate C linical data available since von Recklinghausen's first description of thyrotoxic bone disease, the strong correlation between fracture risk and serum thyroid-stimulating hormone (TSH), recent evidence that TSH receptor (TSHR) polymorphisms are associated with low bone mass, and evidence that bone loss occurs in patients with subclinical hyperthyroidism with normal and low TSH levels all support a role for low TSH in the pathogenesis of hyperthyroid osteoporosis, hitherto attributed solely to high circulating levels of thyroid hormones (1-4).Evidence supporting this premise comes in parallel from genetically manipulated mice lacking receptors for either TSH or thyroid hormones (5, 6). We demonstrated that TSHR deficiency induces a high-turnover osteoporosis, with elevated bone formation and resorption even in TSHR haploinsufficient mice that have normal thyroid function. We found that in ex vivo cell cultures continually exposed to TSH, both osteoblastic bone formation and osteoclastic bone resorption were suppressed. Although these studies point to a primary function for TSH in skeletal homeostasis, data have reestablished a role for the two thyroid hormone receptor (TR) isoforms, TR␣ and TR␤ (6). The deficiency of TR␣ induces osteosclerosis in adult mice, whereas hyperthyroid TR␤-null mice display osteopenia despite high circulating TSH levels (6). Thus, evidence from human and mouse studies underscore the paradigm that both thyroid hormone excess and low TSH levels contribute to hyperthyroid bone loss (7).Hyperthyroidism can arise from adenomatous or inflammatory diseases of the thyroid or from overzealous replacement with thyroid hormones. Hyperthyroidism is Ϸ10-fold more common in women, with an incidence of 1 in 1,000...

Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.

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