Zoledronate facilitates large-scale ex vivo expansion of functional gammadelta T cells from cancer patients for use in adoptive immunotherapy.

BACKGROUND: Human gammadelta T cells can be activated by phospho-antigens and aminobisphosphonates such as zoledronate. Because they can kill tumor cells in a major histocompatibility complex (MHC)-unrestricted manner, adoptive transfer of activated gammadelta T cells may represent a novel cancer immunotherapy. We tested whether gammadelta T cells from advanced cancer patients can be expanded by zoledronate. METHODS: Peripheral blood mononuclear cells from healthy donors and patients with advanced non-small cell lung cancer, bone metastatic breast or prostate cancer, or lung metastatic colorectal cancer, were stimulated with zoledronate (5 microM) and interleukin (IL)-2 (1000 IU/mL) for 14 days. The phenotype and function of the expanded gammadelta T-cell populations from healthy donors and cancer patients were compared. RESULTS: Gammadelta T cells from cancer patients and healthy donors responded to zoledronate equally well in terms of both phenotype and function. gammadelta T cells grew rapidly in vitro and expression of effector molecules, such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, perforin, granzyme B, FasL and TRAIL, increased over time. Cytotoxicity peaked on days 12-14, and proliferation continued up to 14 days, during which time>1x10(9) gammadelta T cells could be obtained from a starting sample of 45-70 mL peripheral blood. DISCUSSION: Using the agent zoledronate, already widely used in the clinic, we have established that efficient large-scale ex vivo expansion of gammadelta T cells from cancer patients is possible. These cells exert potent cytotoxicity and may be used for autologous cellular immunotherapy of cancer.

Na+ dependence of extracellular Ca2+-sensing mechanisms leading to activation of an outwardly rectifying Cl- channel in murine osteoclasts.

An elevation in the extracellular Ca(2+) concentration ([Ca(2+)](o)) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca(2+)](o)-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca(2+)](o)-sensing mechanisms leading to the activation of the Cl(-) channel in murine osteoclasts. A rise in either Ca(2+) or Gd(3+) activated an outwardly rectifying Cl(-) (OR(cl)) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20-30 mmol/L for Ca(2+). Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca(2+)](o)-activated OR(cl) current. Substitution of extracellular Na(+) by an impermeable cation, N-methyl-D-glucamine(+), inhibited the [Ca(2+)](o)-activated OR(cl) channel, suggesting that the activation depended on extracellular Na(+). A blocker for the Na(+)-Ca(2+) exchanger, 2'4'-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca(2+)](o)-activated OR(cl) channel as well. Although 10 mmol/L Ca(2+) activated the OR(cl) current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca(2+)](o)-activated OR(cl) current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na(+)-H(+) exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR(cl) current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca(2+)](o)-sensing mechanisms, leading to activation of the OR(cl) channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na(+)-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na(+) influx.

Apolipoprotein E is found in astrocytes but not in microglia in the normal mouse brain.

Apolipoprotein E (apoE) is a known risk factor for Alzheimer's disease, but neither its roles in the pathogenesis nor its exact physiological functions in the brain is known. In order to study the apoE protein in the brains of normal mouse and transgenic mouse models of neurodegeneration, hamster monoclonal antibodies (MAbs) specific to mouse apoE were generated. N- and C-terminal fragments of mouse apoE protein were produced in E. coli as fusion proteins and used to immunize Armenian hamsters. Specificity of the antibodies was established by immunoblotting against sera and brain homogenates of wild type and apoE-deficient mice. MAb 884F11 was found most suitable for immunohistochemistry on 4% PFA-fixed brain tissues. The strongly positive structure in the normal brain was the astrocytes as identified by simultaneous staining for GFAP with lesser and regionally variable diffuse staining of the neuropil. GFAP-positive cells were variable in their content of apoE. ApoE immunoreactivity in the hippocampus and neocortex did not coincide with the tomato lectin binding, indicating that this apolipoprotein is not detectable in the microglial cells of the normal adult mouse brain.