Christine W. Czarniecki

Phase 3 Trial of Transplantation of Human Islets in Type 1 Diabetes Complicated by Severe Hypoglycemia

OBJECTIVE Impaired awareness of hypoglycemia (IAH) and severe hypoglycemic events (SHEs) cause substantial morbidity and mortality in patients with type 1 diabetes (T1D). Current therapies are effective in preventing SHEs in 50–80% of patients with IAH and SHEs, leaving a substantial number of patients at risk. We evaluated the effectiveness and safety of a standardized human pancreatic islet product in subjects in whom IAH and SHEs persisted despite medical treatment. RESEARCH DESIGN AND METHODS This multicenter, single-arm, phase 3 study of the investigational product purified human pancreatic islets (PHPI) was conducted at eight centers in North America. Forty-eight adults with T1D for >5 years, absent stimulated C-peptide, and documented IAH and SHEs despite expert care were enrolled. Each received immunosuppression and one or more transplants of PHPI, manufactured on-site under good manufacturing practice conditions using a common batch record and standardized lot release criteria and test methods. The primary end point was the achievement of HbA1c <7.0% (53 mmol/mol) at day 365 and freedom from SHEs from day 28 to day 365 after the first transplant. RESULTS The primary end point was successfully met by 87.5% of subjects at 1 year and by 71% at 2 years. The median HbA1c level was 5.6% (38 mmol/mol) at both 1 and 2 years. Hypoglycemia awareness was restored, with highly significant improvements in Clarke and HYPO scores (P > 0.0001). No study-related deaths or disabilities occurred. Five of the enrollees (10.4%) experienced bleeds requiring transfusions (corresponding to 5 of 75 procedures), and two enrollees (4.1%) had infections attributed to immunosuppression. Glomerular filtration rate decreased significantly on immunosuppression, and donor-specific antibodies developed in two patients. CONCLUSIONS Transplanted PHPI provided glycemic control, restoration of hypoglycemia awareness, and protection from SHEs in subjects with intractable IAH and SHEs. Safety events occurred related to the infusion procedure and immunosuppression, including bleeding and decreased renal function. Islet transplantation should be considered for patients with T1D and IAH in whom other, less invasive current treatments have been ineffective in preventing SHEs.

National Institutes of Health–Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities

Eight manufacturing facilities participating in the National Institutes of Health–sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.

Effect of 2′5′-oligoadenylic acid on a mouse cell line partially resistant to interferon

Abstract We have investigated the sensitivity of a mouse cell line, NIH 3T3 (clone 1), that was found to be resistant to the anticellular and certain antiviral activities of interferon, to 2′,5′-oligoadenylate (2′5′A). The 2′5′A was introduced into the cells by coprecipitation with calcium phosphate and by increasing cell permeability with lysolecithin. In both cases neither cellular protein nor DNA synthesis was inhibited by 2′5′A in the NIH 3T3 cells. In contrast, the synthesis of proteins and DNA in L cells, an IFN sensitive line, was inhibited by low concentrations of 2′5′A. Furthermore, treatment of the infected cell cultures with 2′5′A resulted in the inhibition of vesicular stomatitis virus (VSV) replication in L cells but not in NIH 3T3 cells. The production of MuLV in NIH 3T3 cells was also not affected by 2′5′A. These observations, together with our previous finding that NIH 3T3 (clone 1) cells are deficient in RNase F activity suggest that activation of the RNase F by 2′5′A is responsible for the inhibition of both protein and DNA synthesis in sensitive cells.

Ultrastructural analysis of the anti-chlamydial activity of recombinant murine interferon-γ☆

The effect of murine interferon-gamma (MuIFN-gamma) on the developmental cycle of Chlamydia trachomatis in McCoy cells was analyzed by light and electron microscopy. Addition to the culture media of 10 ng/ml of MuIFN-gamma, either 24 hr before or immediately after Chlamydia infection, resulted in a significant inhibition of the growth of this organism. Microscopic analysis showed that with both treatments the majority of microorganisms were arrested at the elementary body stage. Only a few small chlamydial inclusions were detected at 48 hr postinfection and contained predominately reticulate bodies. Furthermore, the growth of Chlamydia was arrested in cells that were treated with MuIFN-gamma at various intervals following infection. Addition of MuIFN-gamma at 8 or 12 hr after infection resulted in the arrest of chlamydial growth before initiation of reticulate body fission. When the MuIFN-gamma was added 24 hr postinfection, we could detect, by electron microscopy, inhibition at the stage of reticulate body replication.

Ultrastructural analysis of the growth cycle of Chlamydia trachomatis in mouse cells treated with recombinant human α-interferons

The effect of two recombinant human hybrid interferons (IFNs), IFN-alpha AD (BglII) and IFN-alpha DA (BglII), on the growth cycle of Chlamydia trachomatis in a murine (McCoy) cell line was investigated. Ultrastructural analysis indicated that IFN-alpha AD inhibited the growth of chlamydia while IFN-alpha DA-treated cells did not significantly differ from the control monolayers. Treatment of the chlamydia-infected monolayers with IFN-alpha AD resulted in an inhibition in the transformation of elementary bodies to reticulate bodies with a consequent marked decrease in the number of chlamydia inclusions. Furthermore, chlamydial inclusions in the IFN-alpha AD-treated cells contained fewer and more immature chlamydial forms than the control or the IFN-alpha DA-treated cells. Secondary infection occurred in the IFN-alpha DA and in the control monolayer, but no such phenomena was detected in the IFN-alpha AD-treated McCoy cells indicating a loss of infectivity of the chlamydial organisms. From this study it can be concluded that purified recombinant human hybrid IFNs may exert an inhibitory effect on the growth cycle of C. trachomatis in a mouse cell line. This inhibition occurs primarily at the point of transformation from elementary to reticulate body.

Disparate response of encephalomyocarditis virus and MM virus to interferon in JLS-V9R cells

JLS-V9R cells, a Balb/c mouse bone marrow cell line chronically infected with Rauscher leukemia virus, were treated with mouse interferon and inoculated with several different lytic viruses. Relatively low interferon concentrations protected the cells against Sindbis virus, vesicular stomatitis virus and MM virus. In contrast, encephalomyocarditis virus replication was inhibited by less than 1 log even with an interferon concentration of 1000 U/ml. These findings provide further evidence that interferon-induced antiviral effects are mediated through multiple mechanisms and demonstrate that even viruses which are classified within the same family (MM and encephalomyocarditis virus) can exhibit differential interferon sensitivities.

Erratum. National Institutes of Health–Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities. Diabetes 2016;65:3418–3428

In the article listed above, Xiaojuan Chen, now of the Columbia Center for Translational Immunology, Columbia University, New York, NY, was erroneously omitted from the author list. Dr. Chen contributed to the Clinical Islet Transplantation (CIT) …