Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance.

The phenomenon of early endotoxin tolerance, which is induced by sublethal injection of lipopolysaccharide (LPS), results in a protracted period of hyporesponsiveness that is most profound at 3 to 4 days after injection and is marked by reduced cytokine production after a challenge injection of LPS. Early endotoxin tolerance is also induced by the nontoxic LPS derivative monophosphoryl lipid A (MPL), although much more of the monophosphoryl derivative is required to produce a state of tolerance equivalent to that evoked by LPS. In this study, equivalent tolerance-inducing doses of LPS and MPL were tested, and the levels of cytokines induced by LPS and MPL were compared. Although induced levels of colony-stimulating factor were comparable following doses of LPS and MPL that elicited an equivalent state of early endotoxin tolerance, levels of tumor necrosis factor, interleukin-6, and interferon were significantly lower in MPL-injected animals. These results suggest that the lowered toxicity of MPL may be related to its elicitation of significantly lower levels of potentially toxic intermediaries such as tumor necrosis factor, interferon, and interleukin-6.     

Natural History of Mouse Syngeneic Lymphoma: Morphologic and Immunologic Aspects

The morphologic changes in lymphoreticular tissues and development of antitumor immune reactions of specific pathogen-free mice injected with syngeneic lymphoma cells were sequentially analyzed. The regional (right inguinal) lymph node demonstrated mild changes indicative of immunologic response. Systemic lymph nodes revealed a moderate degree of immune response on morphologic basis. The spleen was the site of marked activity, characterized by the presence of large pyroninophilic cells and germinal centers. Foci of necrosis in the local tumor accompanied by mature lymphocytes suggested cell-mediated immune rejection. Mice developed circulating antibodies 2 days after implantation. No antibodies were demonstrated attached to fresh tumor cells. Lymphocyte cytotoxic activity was demonstrated beginning on day 4. Both cytotoxic activity and circulating antibodies were no longer detectable after the third week following tumor implantation. Tumor-bearing mice also had an impaired capacity to mount a primary immune reaction to sheep red blood cells. The spleen demonstrated a marked loss of lymphocytes and the subsequent appearance of masses of amyloid material. It is suggested that amyloidosis in lymphoreticular organs is the result of a derangement in the immune response of the host following a prolonged and sustained antigenic stimulation. It appears that in syngeneic pathogen-free mice the spleen plays the major role in immune rejection mechanisms while the draining node only plays a modest role.     

Association of defensin beta-1 gene polymorphisms with asthma

BACKGROUND: Defensins are antimicrobial peptides that may take part in airway inflammation and hyperresponsiveness. OBJECTIVE: We characterized the genetic diversity in the defensin beta-1 (DEFB1) locus and tested for an association between common genetic variants and asthma diagnosis. METHODS: To identify single nucleotide polymorphisms (SNPs), we resequenced this gene in 23 self-defined European Americans and 24 African Americans. To test whether DEFB1 genetic variants are associated with asthma, we genotyped 4 haplotype-tag SNPs in 517 asthmatic and 519 control samples from the Nurses' Health Study (NHS) and performed a case-control association analysis. To replicate these findings, we evaluated the DEFB1 polymorphisms in a second cohort from the Childhood Asthma Management Program. RESULTS: Within the NHS, single SNP testing suggested an association between asthma diagnosis and a 5' genomic SNP (g.-1816 T>C; P = .025) and intronic SNP (IVS+692 G>A; P = .054). A significant association between haplotype (Adenine, Cytosine, Thymine, Adenine [ACTA]) and asthma ( P = .024) was also identified. Associations between asthma diagnosis and both DEFB1 polymorphisms were observed in Childhood Asthma Management Program, a second cohort: g.-1816 T>C and IVS+692 G>A demonstrated significant transmission distortion ( P = .05 and .007, respectively). Transmission distortion was not observed in male subjects. The rare alleles (-1816C and +692A) were undertransmitted to offspring with asthma, suggesting a protective effect, contrary to the findings in the NHS cohort. Similar effects were evident at the haplotype level: ACTA was undertransmitted ( P = .04) and was more prominent in female subjects ( P = .007). CONCLUSION: Variation in DEFB1 contributes to asthma diagnosis, with apparent gender-specific effects.     

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.     

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.     

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.     

Continuous Glucose Monitoring Use in Clinical Trials for On-Market Diabetes Drugs

To the best of our knowledge, there are no published data on the historical and recent use of CGM in clinical trials of pharmacological agents used in the treatment of diabetes. We analyzed 2,032 clinical trials of 40 antihyperglycemic therapies currently on the market with a study start date between 1 January 2000 and 31 December 2019. According to ClinicalTrials.gov, 119 (5.9%) of these trials used CGM. CGM usage in clinical trials has increased over time, rising from <5% before 2005 to 12.5% in 2019. However, it is still low given its inclusion in the American Diabetes Association’s latest guidelines and known limitations of A1C for assessing ongoing diabetes care.

The availability of reliable continuous glucose monitoring (CGM) systems has proven to be a major innovation in diabetes management and research. Most current CGM systems are approved for 7- to 14-day use and use a wire-tipped glucose oxidase sensor inserted in subcutaneous tissue to monitor glucose concentrations in interstitial fluid. One implanted CGM system is approved for longer-term use (90–180 days); it operates with fluorescence-based technology. CGM sensors record a glucose data point every 1–15 minutes (depending on the system), collecting far more granular data and information on glycemic patterns than self-monitoring of blood glucose (SMBG) alone. Real-time CGM or intermittently scanned CGM systems send data continuously or intermittently to dedicated receivers or smartphones, whereas professional CGM systems provide retrospective data, either blinded or unblinded, for analysis and can be used to identify patterns of hypo- and hyperglycemia. Professional CGM can be helpful to evaluate patients when other CGM systems are not available to the patient or the patient prefers a blinded analysis or a shorter experience with unblinded data.In the 20 years since CGM systems first became available to people with diabetes, technological improvements, particularly pertaining to accuracy and form factor, have made CGM increasingly viable for both patient use and clinical investigation (1,2). Average sensor MARD (mean absolute relative difference; a summary accuracy statistic) has decreased from >20 to <10% (3–10), including two systems that do not require fingerstick calibrations and three that are approved to be used for insulin dosing (11). Concurrently, size, weight, and cost of CGM systems have all decreased, while user-friendliness and convenience have increased (12).To encourage use of CGM-derived data, researchers and clinicians have worked to develop a standard set of glycemic metrics beyond A1C. In 2017, two international groups of leading diabetes clinical and research organizations published consensus definitions for key metrics, including clinically relevant glycemic cut points for hypoglycemia (<70 and <54 mg/dL), hyperglycemia (>180 and >250 mg/dL), and time in range (TIR; 70–180 mg/dL) (13,14).CGM-derived metrics provide far greater precision and granularity than is possible with SMBG or A1C data alone (Table 1), enabling clinicians and investigators to better represent inter- and intraday glycemic differences with metrics such as TIR, glycemic variability, and time in hypoglycemia and hyperglycemia (15). Crucially, CGM also allows for the accurate measurement and detection of nocturnal glycemia (16). The use of these metrics enables a more comprehensive understanding of glycemic management that can facilitate individualized treatment for people with diabetes or prediabetes. Although A1C is a useful estimate of mean glucose over the previous 2–3 months, especially when evaluating population health, it is important to include other glycemic outcomes in clinical trials. Furthermore, there is emerging evidence suggesting that TIR predicts the development of microvascular complications at least as well as A1C (17,18).TABLE 1Benefits of CGM Compared With A1C Alone in Assessing Glycemia

Use of re-randomized data in meta-analysis

Background  

Outcomes collected in randomized clinical trials are observations of random variables that should be independent and identically distributed. However, in some trials, the patients are randomized more than once thus violating both of these assumptions. The probability of an event is not always the same when a patient is re-randomized; there is probably a non-zero covariance coming from observations on the same patient. This is of particular importance to the meta-analysts.