Human CXCR1 knock-in mice infer functional expression of a murine ortholog.

Targeting CXCR1 and CXCR2 chemokine receptors to block neutrophil migration to sites of inflammation is a promising therapeutic approach for various inflammatory and autoimmune diseases. However, assessing the translational potential of such therapies using mouse models is challenging due to the unclear expression of CXCR1 at the protein level. Although CXCR2 has been well characterized in both mice and humans, the protein-level expression of CXCR1 in mice (mCXCR1) remains controversial. To address this issue, we generated a novel human CXCR1 knock-in (hCXCR1 KI) mouse model in which the transgene is under the control of the native mouse promoter and regulatory elements. Using an anti-human CXCR1 monoclonal antibody (anti-hCXCR1 monoclonal antibody), we found that hCXCR1 was highly expressed on neutrophils in the hCXCR1 KI mice, comparable to levels observed in human neutrophils. This successful expression of hCXCR1 in this mouse model suggests that functional mCXCR1 likely exists. To investigate the functional role of CXCR1, we investigated how antagonizing this receptor using anti-hCXCR1 monoclonal antibody in the arthritis model would affect disease outcomes. Antibody treatment significantly alleviated all signs of joint inflammation. In summary, our newly generated hCXCR1 KI transgenic mice provide a valuable tool to investigate the therapeutic efficacy of small molecules or monoclonal antibodies that antagonize this receptor in neutrophil-mediated pathologies.

Therapeutic blockade of CXCR2 rapidly clears inflammation in arthritis and atopic dermatitis models: demonstration with surrogate and humanized antibodies.

Neutrophils are the most abundant effector cells of the innate immune system and represent the first line of defense against infection. However, in many common pathologies, including autoimmune diseases, excessive recruitment and activation of neutrophils can drive a chronic inflammatory response leading to unwanted tissue destruction. Several strategies have been investigated to tackle pathologic neutrophil biology, and thus provide a novel therapy for chronic inflammatory diseases. The chemokine receptor CXCR2 plays a crucial role in regulating neutrophil homeostasis and is a promising pharmaceutical target. In this study, we report the discovery and validation of a humanized anti-human CXCR2 monoclonal antibody. To enable in vivo studies, we developed a surrogate anti-mouse CXCR2 antibody, as well as a human knock-in CXCR2 mouse. When administered in models of atopic dermatitis (AD) and rheumatoid arthritis (RA), the antibodies rapidly clear inflammation. Our findings support further developments of anti-CXCR2 mAb approaches not only for RA and AD, but also for other neutrophil-mediated inflammatory conditions where neutrophils are pathogenic and medical needs are unmet.

Selection of a fully human single domain antibody specific to Helicobacter pylori urease.

Helicobacter pylori bacteria are involved in gastroduodenal disorders, including gastric adenocarcinoma. Since the current therapies encounter with some significant shortcomings, much attention has been paid to the development of new alternative diagnostic and treatment modalities such as immunomedicines to target H. pylori. Having used phage display technology, we isolated fully humane small antibody (Ab) fragment (VL) against the Flap region of urease enzyme of H. pylori to suppress its enzymatic activity. Solution biopanning (SPB) and screening process against a customized biotinylated peptide corresponding to the enzyme Flap region resulted in the selection of VL single domain Abs confirmed by the enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. The selected Ab fragments showed a high affinity with a KD value of 97.8 × 10-9 and specificity to the enzyme with high inhibitory impact. For the first time, a VL single domain Ab was isolated by SPB process against a critical segment of H. pylori urease using a diverse semi-synthetic library. Based on our findings, the selected VL Ab fragments can be used for the diagnosis, imaging, targeting, and/or immunotherapy of H. pylori. Further, Flap region shows great potential for vaccine therapy.

Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori.

Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.

Pleiotropic cytotoxicity of VacA toxin in host cells and its impact on immunotherapy.

Introduction: In the recent decades, a number of studies have highlighted the importance of Helicobacter pylori in the initiation and development of peptic ulcer and gastric cancer. Some potential virulence factors (e.g., urease, CagA, VacA, BabA) are exploited by this microorganism, facilitating its persistence through evading human defense mechanisms. Among these toxins and enzymes, vacuolating toxin A (VacA) is of a great importance in the pathogenesis of H. pylori. VacA toxin shows different pattern of cytotoxicity through binding to different cell surface receptors in various cells. Methods: To highlight attempts in treatment for H. pylori infection, here, we discussed the VacA potential as a candidate for development of vaccine and targeted immunotherapy. Furthermore, we reviewed the related literature to provide key insights on association of the genetic variants of VacA with the toxicity of the toxin in cells. Results: A number of investigations on the receptor(s) binding of VacA toxin confirmed the pleiotropic nature of VacA that uses a unique mechanism for internalization through some membrane components such as lipid rafts and glycophosphatidylinositol (GPI)-anchored proteins (GPI-AP). Considering the high potency of VacA toxin in the clinical presentations in infection and assisting persistence and colonization of H. pylori, it is considered as one of the pivotal components in production vaccines and monoclonal antibodies (mAbs). Conclusion: It is possible to generate mAbs with a considerable potential to convert into secretory immunoglobulins that could penetrate into the niche of H. pylori and inhibit its normal functionalities. Further, conjugation of H. pylori targeting Ab fragments with the toxic agents or drug delivery systems (DDSs) offers new generation of H. pylori treatments.

Does L-carnitine improve endothelial function in hemodialysis patients?

BACKGROUND: Atherosclerosis is the leading cause of death in hemodialysis patients. These patients are also very prone to L-carnitine deficiency due to kidney disease. In this clinical trial, we investigated the effect of oral L-carnitine on endothelial function of these patients. MATERIALS AND METHODS: [corrected] We studied 31 adult chronic hemodialysis patients in our center and divided them into two groups. The first group (n = 20) received 1500 mg/dialysis interval (every other day) oral L-carnitine. The control group (n = 11) received placebo for one month. Ultrasonographic measurements of flow mediated dilation and carotid intima-media thickness were performed before and after one month of L-carnitine and placebo therapy. RESULTS: This study showed that after one month of L-carnitine or placebo therapy there was no significant improvement in flow mediated dilation (p = 0.80 and p = 0.59, respectively) or decrease in carotid intima-media thickness (p = 0.12 and p = 0.50, respectively). CONCLUSIONS: Our study revealed that one month of oral L-carnitine therapy did not improve endothelial function in hemodialysis patients. Long-term studies with large sample size using intravenous form and higher doses of the drug are required to clarify the questionable role of L-carnitine in hemodialysis patients.

Complementary and alternative medicine in obstetrics: a survey from Iran.

PURPOSE: This survey assessed the use of complementary and alternative medicine (CAM) methods by obstetricians in the Islamic Republic of Iran. METHODS: Obstetricians in the province of Tehran were identified using the "Ketabe 118 Mashaghel" (2008), a source of medical department information. A survey on the use of CAM methods during childbirth and the reasons behind their application was conducted on site. RESULTS: CAM methods are by in 37.3% (62/166) of the obstetricians. Acupressure, massage, and phytotherapy were found to be the most frequently used methods. Use of CAM was influenced by the employment status of the midwives and inversely correlated with the number of deliveries in the hospital. CONCLUSIONS: CAM methods are used in Iran to some extent. With evidence-based medicine in mind it is interesting to note that in Iran mainly CAM methods which already have some proven benefit are used.