Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130–4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623–630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341–3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

CCL21 is a chemokine that mediates recruitment of multiple leukocyte subsets through CCR7-mediated signaling during the steady state and inflammation. CCL21 plays crucial roles in priming adaptive immunity via governing egress of dendritic cells (DCs) from barrier tissues and T cell entry and positioning in secondary lymphoid organs (1–5). A well-characterized site of CCL21 gradient formation is the skin, where CCL21 is secreted by lymphatic endothelial cells (LECs) and immobilized on extracellular heparan sulfate moieties via interactions with the charged, elongated C-terminal tail of CCL21 (6–8). Here, immobilized CCL21 gradients are essential for interstitial DC trafficking toward lymphatic vessels (LVs) (8), after which CCL21 further contributes to LV attachment (9), infiltration (10), downstream luminal migration (11), and migration from the lymph node (LN) subcapsular sinus (SCS) to the paracortex (12). In-vitro studies have shown that the C-terminal tail of CCL21 can also be proteolytically cleaved by mature DCs to generate solubilized CCL21 (13), with signaling properties distinct from another soluble CCR7 ligand, CCL19 (14, 15). While CCL19 is dispensable for steady-state DC migration (16), important questions regarding the in vivo processing and function of cleaved CCL21 remain.Both forms of CCL21 are also ligands for the atypical chemokine receptor ACKR4 (17), which regulates chemokine bioavailability rather than directly mediating cell migration. ACKR4 expression has been identified in multiple barrier tissues (18–20) and lymphoid tissues (12, 21) where expression is largely restricted to stromal cell populations, with the exception of germinal-center B cells (22). Despite clear evidence of ACKR4 scavenging of CCL21 in vitro, the extent to which it regulates CCL21 in vivo is disputed. We have shown increased CCL21 in the LNs of Ackr4^−/− mice, which was associated with exacerbated Th17 responses in autoimmunity (23) and an ACKR4-dependent increase in CCL21 in tumors that promotes antitumor immunity (24). However, no differences in dermal CCL21 abundance were previously reported in steady-state Ackr4^−/− mice despite steady-state CCR7-dependent DC migratory defects being independent of CCL19 (19), and the contribution of ACKR4 in regulating LN CCL21 abundance has been disputed despite a clear role for ACKR4 in maintaining interfollicular CCL21 gradients in LN (12). These discrepancies have remained unresolved but point to previously unrecognized complexity in ACKR4-dependent regulation of CCL21 in both barrier and lymphoid tissues.     

Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells

Although mature dendritic cells (DCs) are potent initiators of adaptive immune response, immature steady-state DCs contribute to immune tolerance. In this study, we show that ex vivo splenic DCs are capable of inducing conversion of naïve CD4⁺ T cells to adaptive Foxp3⁺CD4⁺ regulatory T cells (aTreg) in the presence of TGF-β. In particular, when compared with splenic CD8α⁻ DCs, the CD8α⁺ DC subset were superior in inducing higher frequencies of conversion. This was not attributable to the difference in basal level of costimulation, because deficiency of CD40 or CD80/86 signaling did not diminish the differential induction of Foxp3. Conversion was regulated by DC maturation status. Further insights into the molecular mechanisms of conversion were gained by analyzing the contribution of several costimulatory and coinhibitory receptors. Costimulatory signals through GITR suppressed conversion, whereas coinhibitory signaling via programmed death 1 ligand (PD-L1) but not PD-L2 was required for conversion. Ex vivo PD-L1^−/− DCs failed to support Foxp3 induction in the presence of TGF-β. In vivo blocking PD-L1 signaling abolished conversion in a tumor-induced aTreg conversion model. Collectively, this study highlights the cellular and molecular parameters that might be exploited to control the de novo generation of aTregs and peripheral tolerance.     

CRF22_01A1 is involved in the emergence of new HIV-1 recombinants in Cameroon

Cameroon is a West African country where high genetic diversity of HIV-1 has been reported. The predominant CRF02_AG is involved in the emergence of more complex intersubtype recombinants. In this study, we sequenced the full-length genome of a novel unique recombinant form of HIV-1, 02CAMLT04 isolated in blood donors in urban Cameroon. Phylogenetic tree and bootscan analysis showed that 02CAMLT04 was complex and seemed to be a secondary recombinant derived from CRF02_AG and CRF22_01A1. The genomic composition of 02CAMLT04 strain showed that it is composed of 3 segments; 24% of the genome is classified as CRF02_AG, spanning most of the envelope gene. The remaining 76% of the genome is classified as CRF22_01A1. In addition, the sequence analysis of 13 full-length sequences from HIV-1-positive specimens received from Cameroon between 2002 and 2010 indicated that 5 specimens are pure CRF22_01A1 viruses, and 6 others have homology with CRF22_01A1 sequences in either gag, pol, or env region, whereas 6% of strains contain portions of CRF22_01A1. Further study demonstrated that CRF22_01A1 is a primary prevalence strain co-circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2), and CRFs (CRF01_AE or CRF02_AG). Our studies show that novel recombinants between CRF22_01A1 and other clades and recombinant forms may be emerging in Cameroon that could contribute to the future global diversity of HIV-1 in this region and worldwide.     

Are lasers superior to lights in the photoepilation of Fitzpatrick V and VI skin types? – A comparison between Nd:YAG laser and intense pulsed light

Background and objectives: There are no large volume comparative studies available to compare the efficacy of lasers over lights for hair removal in Fitzpatrick V and VI skin types. This study is designed to compare the efficacy of Nd:YAG laser versus IPL in the darker skin types. Study design/materials and methods: Thirty-nine patients included in Group-1 were treated with Nd:YAG and 31 in Group-2 with IPL. Both groups received 5 sessions of treatment. The hair counts were assessed using digital photography and manual counting method before and after treatment and the results were analysed. Patient satisfaction scores and pain scores were recorded in each session and compared. Results: Mean hair reduction in the IPL group was 25.70 and Nd:YAG group was 24.12 (95% CI). In the Nd:YAG group, 59% of subjects had burning sensation while the figure was 32.3% in IPL group. Burning was less in IPL group (p < 0.023). There were no statistically significant differences noticed regarding hyperpigmentation in both the groups (p < 0.115). Conclusion: Both Nd:YAG and IPL are equally effective for epilation of the darker skin types. Nd:YAG is associated with mild burning sensation in a significant number of patients. Patient satisfaction scores were comparable in both the groups.     

Association of genetic polymorphism of glutathione S-transferase M1, T1, P1 and susceptibility to bladder cancer

OBJECTIVE: Glutathione-S-transferases (GSTs) are active in the detoxification of wide variety of endogenous or exogenous carcinogens. We examined the association of the GST gene polymorphism with sporadic bladder cancer patients in Northern India. MATERIAL AND METHODS: The study constituted of 106 bladder cancer cases and 370 age-matched controls. The GSTT1 and GSTM1 null genotypes were identified by multiplex PCR and GSTP1313 A/G by Polymerase Chain Reaction/Restriction Fragment Length Polymorphism method (PCR/RFLP). RESULTS: We observed non-significant association in null alleles of the GSTM1 (p = 0.611, OR = 1.12, 95% CI = 0.72-1.74 and GSTT1 (p = 0.135, OR = 1.45, 95% CI = 0.89-2.37) with risk of bladder cancer. However, the G/G genotype of the GSTP1 gene polymorphism was highly significant when compared to controls (p=0.000, OR = 7.12, 95% CI = 3.14-16.16). The combined analysis of the three risk genotypes demonstrated further increase in the risk of bladder cancer (p = 0.000, OR = 7.29 95% CI = 2.81-18.93). CONCLUSION: Our study demonstrated that GSTP1313 G/G polymorphism is a strong predisposing risk factor for bladder cancer. Combination of three GST genotypes association exhibiting gene-gene interaction further substantiates the increased risk of bladder cancer.     

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol‐9

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62‐kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol‐9 (N‐9) in vitro. The sperm immobilisation studies showed that 100 μg ml^?1 of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo‐osmotic swelling in Rp and N‐9 treated groups in comparison with the control. In Rp and N‐9 treated groups, the number of acrosome‐reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5′‐nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.     

Preservation of the antioxidant status in chemically-induced diabetes mellitus by melatonin

Oxidant stress is believed to be enhanced in patients with diabetes mellitus, which may lead to endothelial dysfunction and the development of atherosclerosis. In diabetes, hyperglycemia drives non-enzymatic glycation and oxidation of proteins and lipids which enhances the formation of advanced glycation end products (AGEs), which may be involved in the pathogenesis of diabetic vascular disease. The macrovascular complications of diabetes seem to be due to enhanced cellular oxidant stress by the interaction of AGEs with their receptor. It would be worthwhile to devise methods to reduce this oxidant stress. In alloxan-induced diabetic rats lipid peroxidation products were increased, while levels of nitric oxide glutathione peroxidase and superoxide dismutase were reduced. Melatonin restored these biochemical abnormalities to normalcy independent of hyperglycemia. This model can be used to study the role of oxidant stress in the development of macrovascular complications in diabetes mellitus.     

Symphysis-fundal height measurement--a reliable parameter for assessment of fetal growth

Intrauterine growth retardation (IUGR) is one of the major causes of perinatal mortality in countries like India. Fundal height traditionally measured in relation to umbilicus and xiphisternum is of little value in predicting the fetal growth. Some workers have found that symphysis fundal height (SFH) measurements could be useful in screening pregnancies for growth retardation. A prospective study was taken up in 109 pregnant women attending the antenatal clinic of our Institution. Serial measurements of SFH, abdominal girth, double abdominal wall thickness (DAWT) and maternal weight gain were recorded. SFH measurements obtained were arranged on the basis of 10th, 50th and 90th percentile and represented graphically. Statistical analysis showed that the coefficient of variation was smallest for SFH as compared to abdominal girth and maternal weight gain. The babies (single born) delivered were between 2600 g and 3700 g irrespective of whether the maternal weight gain was 143 g/week or 424 g/week. The abdominal wall thickness had no influence on the measurement of SFH. An attempt was made to develop a nomogram of SFH for our population. This is a simple, reliable and inexpensive method in the screening of pregnancies for IUGR.     

Antibodies to Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rickettsiae among febrile patients in rural areas of Malaysia

A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.